Bcl-2 inhibitors

ABSTRACT

The disclosure includes compounds of Formula (I) 
     
       
         
         
             
             
         
       
         
         
           
             wherein W, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , m, n, L, and Z 1 , are defined herein. Also disclosed is a method for treating a neoplastic disease and autoimmune disease with these compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/045,736, filed on Jul. 26, 2018, which is a continuation ofInternational Patent Application No. PCT/US2017/015281, filed on Jan.27, 2017, which claims the benefit of the filing date of U.S.Provisional Application No. 62/289,209, filed on Jan. 30, 2016. Theentire contents of each of the aforementioned applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a conserved and regulatedprocess that is the primary mechanism for the removal of aged, damagedand unnecessary cells. The ability to block apoptotic signaling is a keyhallmark of cancer and is thus important for oncogenesis, tumormaintenance and chemoresistance Manahan, D. & Weinberg, R. A. Thehallmarks of cancer. Cell 100, 57-70 (2000)1. Dynamic bindinginteractions between prodeath (for example, BCL-2associated X protein(BAX), BCL-2 antagonist/killer 1 (BAK), BCL-2associated agonist of celldeath (BAD), BCL-2like 11 (BIM), NOXA and BCL-2 binding component 3(PUMA)) and prosurvival (BCL-2, BCL-XL, BCL-2like 2 (BCL-W), myeloidcell leukemia sequence 1 (MCL-1) and BCL-2related protein Al (BFL-1))proteins in the BCL-2 family control commitment to programmed celldeath. Altering the balance among these opposing factions provides onemeans by which cancer cells undermine normal apoptosis and gain asurvival advantage [Youle, R. J. & Strasser, A. The BCL-2 protein familyopposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol.9, 47-59 (2008)].

BCL-2, the first identified apoptotic regulator, was originally clonedfrom the breakpoint of a t(14;18) translocation present in human B celllymphomas[Tsujimoto, Y., et al. Science 228, 1440-1443 (1985); Cleary,M. L., et al Cell 47, 19-28 (1986); Boise, L. H. et al. Cell 74, 597-608(1993)]. This protein has since been shown to have a dominant role inthe survival of multiple lymphoid malignancies [Vaux, D. L., et al pre-Bcells. Nature 335, 440-442 (1988)]. Overexpression of Bcl-2 proteinscorrelates with resistance to chemotherapy, clinical outcome, diseaseprogression, overall prognosis or a combination thereof in variouscancers and disorders of the immune system. Involvement of Bcl-2proteins in bladder cancer, brain cancer, breast cancer, bone marrowcancer, cervical cancer, chronic lymphocytic leukemia, colorectalcancer, esophageal cancer, hepatocellular cancer, lymphoblasticleukemia, follicular lymphoma, lymphoid malignancies of T-cell or B-cellorigin, melanoma, myelogenous leukemia, myeloma, oral cancer, ovariancancer, non-small cell lung cancer, prostate cancer, small cell lungcancer, spleen cancer, and the like is described in commonly-ownedPCT/US2004/36770, published as WO 2005/049593, and PCT/US/2004/37911,published as WO 2005/024636. Involvement of Bcl-2 proteins in immune andautoimmune diseases is described in Current Allergy and Asthma Reports2003, 3, 378-384; British Journal of Hematology 2000, 110(3), 584-90;Blood 2000, 95(4), 1283-92; and New England Journal of Medicine 2004,351(14), 1409-1418. Involvement of Bcl-2 proteins in arthritis isdisclosed in commonly-owned U.S. Provisional Patent Application Ser. No.60/988,479. Involvement of Bcl-2 proteins in bone marrow transplantrejection is disclosed in commonly-owned U.S. patent application Ser.No. 11/941,196.

In the last decade, several Bcl-2 inhibitors such as ABT-737, ABT-263,and ABT-199 as shown below have been identified and entered humanclinical trials for cancers treatment.

ABT-737 is discovered by nuclear magnetic resonance (NMR)-basedscreening, parallel synthesis and structure based fragment drug design[Tilman Oltersdorf, et al, Nature, Vol 435, 2005, p 677]. ABT-737 asmall-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-XLand Bcl-w, with an affinity two to three orders of magnitude more potentthan previously reported compounds. Mechanistic studies reveal thatABT-737 does not directly initiate the apoptotic process, but enhancesthe effects of death signals, displaying synergistic cytotoxicity withchemotherapeutics and radiation. ABT-737 exhibitssingle-agent-mechanism-based killing of cells from lymphoma andsmall-cell lung carcinoma lines, as well as primary patient-derivedcells, and in animal models, ABT-737 improves survival, causesregression of established tumors, and produces cures in a highpercentage of the mice. Unfortunately, ABT-737 is not orallybioavailable and its low aqueous solubility makes formulation forintravenous delivery extremely difficult.

After extensive MedChem effort, an orally bioavailable Bcl-2 inhibitorABT-263 (Navitoclax) has been developed [Cheol-Min Park, et al J. Med.Chem. 2008, 51, 6902-6915]. ABT-263 is a potent inhibitor of Bcl-xL,Bcl-2 and Bcl-w with Ki of ≤0.5 nM, ≤nM and ≤1 nM. ABT-263 has a IC₅₀ of110 nM against SCLC H146 cell line, When ABT-263 is administered at 100mg/kg/day in the H345 xenograft model, significant antitumor efficacy isobserved with 80% TGI and 20% of treated tumors indicating at least a50% reduction in tumor volume. Oral administration of ABT-263 alonecauses complete tumor regressions in xenograft models of small-cell lungcancer and acute lymphoblastic leukemia [Tse C, et al. Cancer Res. 2008,68(9), 3421-3428]. Unfortunately, in the clinical trial, the inhibitionof BCL-XL by ABT-263 (navitoclax) induces a rapid,concentration-dependent decrease in the number of circulating platelets.This mechanism-based thrombocytopenia is the dose-limiting toxicity ofsingle-agent navitoclax treatment in patients and limits the ability todrive drug concentrations into a highly efficacious range.

Thus, a BCL-2 selective (BCL-XL sparing) inhibitor would culminate insubstantially reduced thrombocytopenia while maintaining efficacy inlymphoid malignancies. The resulting increase in the therapeutic windowshould allow for greater BCL-2 suppression and clinical efficacy inBCL-2dependent tumor types, After extensive MedChem, ABT-199 (GDC-0199)has been successfully developed[Andrew J Souers, et al, Nature Medicine,Volume 19, 22, p202, 2013]. ABT-199 is a Bcl-2-selective inhibitor withKi of <0.01 nM, >4800-fold more selective versus Bcl-xL and Bcl-w, andno activity to Mc1-1. ABT-199 potently inhibits RS4; 11 cells with EC₅₀of 8 nM. In addition, ABT-199 induces a rapid apoptosis in RS4; 11 cellswith cytochrome c release, caspase activation, and the accumulation ofsub-G0/G1 DNA. Quantitative immunoblotting reveals that sensitivity toABT-199 correlated strongly with the expression of Bcl-2, including NHL,DLBCL, MCL, AML and ALL cell lines. ABT-199 also induces apoptosis inCLL with an average EC₅₀ of 3.0 nM. A single dose of 100 mg/kg ofABT-199 causes a maximal tumor growth inhibition of 95% and tumor growthdelay of 152% in RS4; 11 xenografts. ABT-199 also inhibits xenograftgrowth (DoHH2, Granta-519) as a single agent or in combination withBendamustine and other agents. Human Phase I and II data showed thatABT-199 is highly efficacious for CLL, however, ABT⁻199 causes lethaltumour lysis syndrome in which the debris of dying cancer cellsoverwhelms the kidney and other organs.

Although the Bcl-2 inhibitors, particularly the Bcl-2 selectiveinhibitor ABT-199 have made a significant contribution to the art, thereis a continuing search in this field of art for improvedpharmaceuticals.

SUMMARY OF THE INVENTION

In a first embodiment, this invention provides compounds of the Formula(I) or an N-oxide thereof, or a pharmaceutically acceptable salt,solvate, polymorph, tautomer, stereoisomer, an isotopic form, or aprodrug of said compound of Formula (I) or N-oxide thereof:

wherein

Z₁ is absent, (CH₂)_(p), N(H), O, S, C(O), S(O)₂, OC(O), C(O)O, OS(O)₂,S(O)₂O, C(O)S, SC(O), C(O)C(O), C(O)N(H), N(H)C(O), S(O)₂N(H),N(H)S(O)₂, OC(O)O, OC(O)S, OC(O)N(H), N(H)C(O)O, N(H)C(O)S,N(H)C(O)N(H), (CH₂)_(p)N(H)(CH₂)_(q), (CH₂)_(p)N(H)C(O)(CH₂)_(q),(CH₂)_(p)C(O)N(H)(CH₂)_(q), OC(O)N(H)(CH₂)_(p+1)N(H)(CH₂)_(q), abivalent alkenyl group, or a bivalent alkynyl group;

W is CH or N, and the tricylic

is optionally substituted with one or more R₇;

each of R₁, R₂, R₃, R₄, R₅, R₆, and R₇, independently, is H, alkyl,alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aryl, heteroaryl, halo, nitro, oxo, cyano, OR_(a),SR_(a), alkyl-R_(a), NH(CH₂)_(p)R_(a), C(O)R_(a), S(O)R_(a), SO₂R_(a),C(O)OR_(a), OC(O)R_(a), NR_(b)R_(c), P(O)R_(b)R_(c), C(O)N(R_(b))R_(c),N(R_(b))C(O)R_(c), SO₂N(R_(b))R_(c), or N(R_(b))SO₂R_(c), in which saidcycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,heteroaryl is optionally substituted with one or more R_(d);

optionally, two of R₂ group, taken together with the atom to which theyare attached, may optionally form a cycloalkyl or heterocycloalkyloptionally substituted with one or more R_(d);

R_(a), R_(b), R_(c) and R_(d), independently, is H, alkyl, alkenyl,alkynyl, halo, cyano, amine, nitro, hydroxy, C(O)NHOH, alkoxy,alkoxyalkyl, haloalkyl, hydroxyalkyl, aminoalkyl, alkylcarbonyl,alkoxycarbonyl, alkylcarbonylamino, alkylamino, oxo, halo-alkylamino,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, orheteroaryl;

each of R₁, R₂, independently, is H or alkyl; or two of R₂ group, takentogether with the atom to which they are attached, may optionally form acycloalkyl;

L is absent, alkylene, alkenylene, alkynylene, cycloalkylene,cycloalkenylene, heterocycloalkylene, heterocycloalkenylene, whereinsaid alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene,heterocycloalkylene, or heterocycloalkenylene is optionally substitutedwith one or more R_(d); and

each of m, p, and q, independently, is 0, 1, 2, 3, or 4.

In a second embodiment, the invention provides the compounds accordingto the previous embodiment, wherein the compound is represented byFormula (II):

wherein the tricylic

is unsubstituted, wherein the reminder of the variables are as definedin the first embodiment.

In a third embodiment, the invention provides the compounds according tothe previous embodiments, wherein the compound is represented by Formula(III)

wherein the reminder of the variables are as defined in the firstembodiment.

In a fourth embodiment, the invention provides the compounds accordingto the previous embodiments, wherein R₁ is halo (e.g., Cl), wherein thereminder of the variables are as defined in the first, second or thirdembodiment.

In a fifth embodiment, the invention provides the compounds according tothe previous embodiments, wherein R₅ independently, is nitro, halo, orSO₂R_(a), wherein the reminder of the variables are as defined in thefirst, second, third or fourth embodiment.

In a sixth embodiment, the invention provides the compounds according tothe previous embodiments, wherein Z₁ is absent, NH, O, or S, wherein thereminder of the variables are as defined in the first, second, third,fourth, or fifth embodiment. In a seventh embodiment, the inventionprovides the compounds according to the previous embodiments, wherein Lis absent or C₁₋₃alkylene, wherein the reminder of the variables are asdefined in the first, second, third, fourth, fifth, or sixth embodiment.

In an eighth embodiment, the invention provides the compounds accordingto the previous embodiments, wherein R₆ is H, 4-6 membered cycloalkyl or4-6 membered heterocyclyl, wherein the 4-6 membered cycloalkyl or 4-6membered heterocyclyl is optionally substituted with one or moresubstituents selected from the group consisting of halo, C₁₋₄alkyl, CN,—OH, C₁₋₄alkoxy, —S(O)₂CH₃, —COCH₃, 3-6 membered cycloalkyl, and 3-6membered heterocyclyl, wherein the reminder of the variables are asdefined in the first, second, third, fourth, fifth, sixth, or seventhembodiment

Compounds of the invention may contain one or more asymmetric carbonatoms. Accordingly, the compounds may exist as diastereomers,enantiomers, or mixtures thereof. Each of the asymmetric carbon atomsmay be in the R or S configuration, and both of these configurations arewithin the scope of the invention.

A modified compound of any one of such compounds including amodification having an improved (e.g., enhanced, greater) pharmaceuticalsolubility, stability, bioavailability, and/or therapeutic index ascompared to the unmodified compound is also contemplated. Exemplarymodifications include (but are not limited to) applicable prodrugderivatives, and deuterium-enriched compounds.

It should be recognized that the compounds of the present invention maybe present and optionally administered in the form of salts or solvates.The invention encompasses any pharmaceutically acceptable salts andsolvates of any one of the above-described compounds and modificationsthereof.

Also within the scope of this invention is a pharmaceutical compositioncontaining one or more of the compounds, modifications, and/or salts andthereof described above for use in treating a neoplastic disease,therapeutic uses thereof, and use of the compounds for the manufactureof a medicament for treating the disease/disorder.

This invention also relates to a method of treating a neoplastic diseaseor an autoimmune disease by administering to a subject in need thereofan effective amount of one or more of the compounds, modifications,and/or salts, and compositions thereof described above. The neoplasticdisease or the autoimmune disease can be ameliorated by inhibition ofbcl-2. In one embodiment, the neoplastic disease is leukemia, lymphoma,or multiple myeloma.

Also provided herein is the use of one or more of the disclosedcompounds, or a pharmaceutically acceptable salt thereof, or thepharmaceutical composition thereof, for the preparation of a medicamentfor the treatment of a neoplastic disease or an autoimmune disease.

In another embodiment provided herein, the disclosed compounds, or apharmaceutically acceptable salt thereof, or the pharmaceuticalcomposition thereof are for use in treating a neoplastic disease or anautoimmune disease.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and from the claims. Itshould be understood that all embodiments/features of the invention(compounds, pharmaceutical compositions, methods of make/use, etc)described herein, including any specific features described in theexamples and original claims, can combine with one another unless notapplicable or explicitly disclaimed.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary compounds described herein include, but are not limited to,the following:

Compounds of the invention may contain one or more asymmetric carbonatoms. Accordingly, the compounds may exist as diastereomers,enantiomers or mixtures thereof. The syntheses of the compounds mayemploy racemates, diastereomers or enantiomers as starting materials oras intermediates. Diastereomeric compounds may be separated bychromatographic or crystallization methods. Similarly, enantiomericmixtures may be separated using the same techniques or others known inthe art. Each of the asymmetric carbon atoms may be in the R or Sconfiguration and both of these configurations are within the scope ofthe invention.

A modified compound of any one of such compounds including amodification having an improved (e.g., enhanced, greater) pharmaceuticalsolubility, stability, bioavailability and/or therapeutic index ascompared to the unmodified compound is also contemplated. The examplesof modifications include but not limited to the prodrug derivatives, andthe deuterium-enriched compounds. For example:

-   -   Prodrug derivatives: prodrugs, upon administration to a subject,        will converted in vivo into active compounds of the present        invention [Nature Reviews of Drug Discovery, 2008, Volume 7,        p255]. It is noted that in many instances, the prodrugs        themselves also fall within the scope of the range of compounds        according to the present invention. The prodrugs of the        compounds of the present invention can be prepared by standard        organic reaction, for example, by reacting with a carbamylating        agent (e.g., 1,1-acyloxyalkylcarbonochloridate, para-nitrophenyl        carbonate, or the like) or an acylating agent. Further examples        of methods and strategies of making prodrugs are described in        Bioorganic and Medicinal Chemistry Letters, 1994, Vol. 4, p.        1985.    -   Deuterium-enriched compounds: deuterium (D or ²H) is H) a        stable, non-radioactive isotope of hydrogen and has an atomic        weight of 2.0144. Hydrogen naturally occurs as a mixture of the        isotopes ^(X)H (hydrogen or protium), D (²H or deuterium), and T        (³H or tritium). The natural abundance of deuterium is 0.015%.        One of ordinary skill in the art recognizes that in all chemical        compounds with a H atom, the H atom actually represents a        mixture of H and D, with about 0.015% being D. Thus, compounds        with a level of deuterium that has been enriched to be greater        than its natural abundance of 0.015%, should be considered        unnatural and, as a result, novel over their non-enriched        counterparts.

It should be recognized that the compounds of the present invention maybe present and optionally administered in the form of salts, andsolvates. For example, it is within the scope of the present inventionto convert the compounds of the present invention into and use them inthe form of their pharmaceutically acceptable salts derived from variousorganic and inorganic acids and bases in accordance with procedures wellknown in the art.

When the compounds of the present invention possess a free base form,the compounds can be prepared as a pharmaceutically acceptable acidaddition salt by reacting the free base form of the compound with apharmaceutically acceptable inorganic or organic acid, e.g.,hydrohalides such as hydrochloride, hydrobromide, hydroiodide; othermineral acids such as sulfate, nitrate, phosphate, etc.; and alkyl andmonoarylsulfonates such as ethanesulfonate, toluenesulfonate andbenzenesulfonate; and other organic acids and their corresponding saltssuch as acetate, tartrate, maleate, succinate, citrate, benzoate,salicylate and ascorbate. Further acid addition salts of the presentinvention include, but are not limited to: adipate, alginate, arginate,aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate,camphorsulfonate, caprylate, chloride, chlorobenzoate,cyclopentanepropionate, digluconate, dihydrogenphosphate,dinitrobenzoate, dodecylsulfate, fumarate, galacterate (from mucicacid), galacturonate, glucoheptaoate, gluconate, glutamate,glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate,hippurate, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate,lactate, lactobionate, malonate, mandelate, metaphosphate,methanesulfonate, methylbenzoate, monohydrogenphosphate,2-naphthalenesulfonate, nicotinate, oxalate, oleate, pamoate, pectinate,persulfate, phenylacetate, 3-phenylpropionate, phosphonate andphthalate. It should be recognized that the free base forms willtypically differ from their respective salt forms somewhat in physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free base forms for the purposes ofthe present invention.

When the compounds of the present invention possess a free acid form, apharmaceutically acceptable base addition salt can be prepared byreacting the free acid form of the compound with a pharmaceuticallyacceptable inorganic or organic base. Examples of such bases are alkalimetal hydroxides including potassium, sodium and lithium hydroxides;alkaline earth metal hydroxides such as barium and calcium hydroxides;alkali metal alkoxides, e.g., potassium ethanolate and sodiumpropanolate; and various organic bases such as ammonium hydroxide,piperidine, diethanolamine and N-methylglutamine Also included are thealuminum salts of the compounds of the present invention. Further basesalts of the present invention include, but are not limited to: copper,ferric, ferrous, lithium, magnesium, manganic, manganous, potassium,sodium and zinc salts. Organic base salts include, but are not limitedto, salts of primary, secondary and tertiary amines, substituted aminesincluding naturally occurring substituted amines, cyclic amines andbasic ion exchange resins, e.g., arginine, betaine, caffeine,chloroprocaine, choline, N,N′-dibenzylethylenediamine (benzathine),dicyclohexylamine, diethanolamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine,hydrabamine, iso-propylamine, lidocaine, lysine, meglumine,N-methyl-D-glucamine, morpholine, piperazine, piperidine, polyamineresins, procaine, purines, theobromine, triethanolamine, triethylamine,trimethylamine, tripropylamine and tris-(hydroxymethyl)-methylamine(tromethamine) It should be recognized that the free acid forms willtypically differ from their respective salt forms somewhat in physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid forms for the purposes ofthe present invention.

In one aspect, a pharmaceutically acceptable salt is a hydrochloridesalt, hydrobromide salt, methanesulfonate, toluenesulfonate, acetate,fumarate, sulfate, bisulfate, succinate, citrate, phosphate, maleate,nitrate, tartrate, benzoate, biocarbonate, carbonate, sodium hydroxidesalt, calcium hydroxide salt, potassium hydroxide salt, tromethaminesalt, or mixtures thereof.

Compounds of the present invention that comprise tertiarynitrogen-containing groups may be quaternized with such agents as (C₁₋₄)alkyl halides, e.g., methyl, ethyl, iso-propyl and tert-butyl chlorides,bromides and iodides; di-(C₁-₄) alkyl sulfates, e.g., dimethyl, diethyland diamyl sulfates; alkyl halides, e.g., decyl, dodecyl, lauryl,myristyl and stearyl chlorides, bromides and iodides; and aryl (C₁-₄)alkyl halides, e.g., benzyl chloride and phenethyl bromide. Such saltspermit the preparation of both water- and oil-soluble compounds of theinvention.

Amine oxides, also known as amine-N-oxide and N-oxide, of anti-canceragents with tertiary nitrogen atoms have been developed as prodrugs [MolCancer Therapy. 2004 March; 3(3):233-44]. Compounds of the presentinvention that comprise tertiary nitrogen atoms may be oxidized by suchagents as hydrogen peroxide (H₂O₂), Caro's acid or peracids likemeta-Chloroperoxybenzoic acid (mCPBA) to from amine oxide.

The invention encompasses pharmaceutical compositions comprising thecompound of the present invention and pharmaceutical excipients, as wellas other conventional pharmaceutically inactive agents. Any inertexcipient that is commonly used as a carrier or diluent may be used incompositions of the present invention, such as sugars, polyalcohols,soluble polymers, salts and lipids. Sugars and polyalcohols which may beemployed include, without limitation, lactose, sucrose, mannitol, andsorbitol. Illustrative of the soluble polymers which may be employed arepolyoxyethylene, poloxamers, polyvinylpyrrolidone, and dextran. Usefulsalts include, without limitation, sodium chloride, magnesium chloride,and calcium chloride. Lipids which may be employed include, withoutlimitation, fatty acids, glycerol fatty acid esters, glycolipids, andphospholipids.

In addition, the pharmaceutical compositions may further comprisebinders (e.g., acacia, cornstarch, gelatin, carbomer, ethyl cellulose,guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose,povidone), disintegrating agents (e.g., cornstarch, potato starch,alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guargum, sodium starch glycolate, Primogel), buffers (e.g., tris-HCL,acetate, phosphate) of various pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors,surfactants (e.g., sodium lauryl sulfate), permeation enhancers,solubilizing agents (e.g., glycerol, polyethylene glycerol,cyclodextrins), a glidant (e.g., colloidal silicon dioxide),anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylatedhydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose,hydroxypropylmethyl cellulose), viscosity increasing agents (e.g.,carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum),sweeteners (e.g., sucrose, aspartame, citric acid), flavoring agents(e.g., peppermint, methyl salicylate, or orange flavoring),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g., stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g., colloidal silicon dioxide),plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers(e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate, methylcellulose, hydroxyethyl cellulose, carboxymethylcellulose sodium),polymer coatings (e.g., poloxamers or poloxamines), coating and filmforming agents (e.g., ethyl cellulose, acrylates, polymethacrylates)and/or adjuvants.

In one embodiment, the pharmaceutical compositions are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

Additionally, the invention encompasses pharmaceutical compositionscomprising any solid or liquid physical form of the compound of theinvention. For example, the compounds can be in a crystalline form, inamorphous form, and have any particle size. The particles may bemicronized, or may be agglomerated, particulate granules, powders, oils,oily suspensions or any other form of solid or liquid physical form.

When compounds according to the present invention exhibit insufficientsolubility, methods for solubilizing the compounds may be used. Suchmethods are known to those of skill in this art, and include, but arenot limited to, pH adjustment and salt formation, using co-solvents,such as ethanol, propylene glycol, polyethylene glycol (PEG) 300, PEG400, DMA (10-30%), DMSO (10-20%), NMP (10-20%), using surfactants, suchas polysorbate 80, polysorbate 20 (1-10%), cremophor EL, Cremophor RH40,Cremophor RH60 (5-10%), Pluronic F68/Poloxamer 188 (20-50%), SolutolHS15 (20-50%), Vitamin E TPGS, and d-α-tocopheryl PEG 1000 succinate(20-50%), using complexation such as HPPCD and SBEr3CD (10-40%), andusing advanced approaches such as micelle, addition of a polymer,nanoparticle suspensions, and liposome formation.

A wide variety of administration methods may be used in conjunction withthe compounds of the present invention. Compounds of the presentinvention may be administered or coadministered orally, parenterally,intraperitoneally, intravenously, intraarterially, transdermally,sublingually, intramuscularly, rectally, transbuccally, intranasally,liposomally, via inhalation, vaginally, intraoccularly, via localdelivery (for example by catheter or stent), subcutaneously,intraadiposally, intraarticularly, or intrathecally. The compoundsaccording to the invention may also be administered or coadministered inslow release dosage forms. Compounds may be in gaseous, liquid,semi-liquid or solid form, formulated in a manner suitable for the routeof administration to be used. For oral administration, suitable solidoral formulations include tablets, capsules, pills, granules, pellets,sachets and effervescent, powders, and the like. Suitable liquid oralformulations include solutions, suspensions, dispersions, emulsions,oils and the like. For parenteral administration, reconstitution of alyophilized powder is typically used.

As used herein, “acyl” means a carbonyl containing substituentrepresented by the formula —C(O)—R in which R is H, alkyl, a carbocycle,a heterocycle, carbocycle-substituted alkyl or heterocycle-substitutedalkyl wherein the alkyl, alkoxy, carbocycle and heterocycle are asdefined herein. Acyl groups include alkanoyl (e.g. acetyl), aroyl (e.g.benzoyl), and heteroaroyl.

“Aliphatic” means a moiety characterized by a straight or branched chainarrangement of constituent carbon atoms and may be saturated orpartially unsaturated with one or more double or triple bonds.

The term “alkyl” refers to a straight or branched hydrocarbon containing1-20 carbon atoms (e.g., C₁-C₁₀). Examples of alkyl include, but are notlimited to, methyl, methylene, ethyl, ethylene, n-propyl, i-propyl,n-butyl, i-butyl, and t-butyl. Preferably, the alkyl group has one toten carbon atoms. More preferably, the alkyl group has one to fourcarbon atoms.

The term “alkenyl” refers to a straight or branched hydrocarboncontaining 2-20 carbon atoms (e.g., C₂-C₁₀) and one or more doublebonds. Examples of alkenyl include, but are not limited to, ethenyl,propenyl, and allyl. Preferably, the alkylene group has two to tencarbon atoms. More preferably, the alkylene group has two to four carbonatoms.

The term “alkynyl” refers to a straight or branched hydrocarboncontaining 2-20 carbon atoms (e.g., C₂-C₁₀) and one or more triplebonds. Examples of alkynyl include, but are not limited to, ethynyl,1-propynyl, 1- and 2-butynyl, and 1-methyl-2-butynyl. Preferably, thealkynyl group has two to ten carbon atoms. More preferably, the alkynylgroup has two to four carbon atoms.

The term “alkylamino” refers to an N(R)-alkyl in which R can be H,alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,heterocycloalkenyl, aryl, or heteroaryl.

“Alkoxy” means an oxygen moiety having a further alkyl substituent.

“Alkoxycarbonyl” means an alkoxy group attached to a carbonyl group.

“Oxoalkyl” means an alkyl, further substituted with a carbonyl group.The carbonyl group may be an aldehyde, ketone, ester, amide, acid oracid chloride.

The term “cycloalkyl” refers to a saturated hydrocarbon ring systemhaving 3 to 30 carbon atoms (e.g., C₃-C₁₂, C₃-C₈, C₃-C₆). Examples ofcycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The term“cycloalkenyl” refers to a non-aromatic hydrocarbon ring system having 3to 30 carbons (e.g., C₃-C₁₂) and one or more double bonds. Examplesinclude cyclopentenyl, cyclohexenyl, and cycloheptenyl.

The term “heterocycloalkyl” refers to a nonaromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having one or more heteroatoms (such as O, N, S, B, P, Si, orSe). Examples of heterocycloalkyl groups include, but are not limitedto, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, andtetrahydrofuranyl.

The term “heterocycloalkenyl” refers to a nonaromatic 5-8 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having one or more heteroatoms (such as O, N, S, P, B, Si, or Se)and one or more double bonds.

The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic,14-carbon tricyclic aromatic ring system. Examples of aryl groupsinclude, but are not limited to, phenyl, naphthyl, and anthracenyl. Theterm “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12membered bicyclic, or 11-14 membered tricyclic ring system having one ormore heteroatoms (such as O, N, S, P, or Se). Examples of heteroarylgroups include pyridyl, furyl, imidazolyl, benzimidazolyl, pyrimidinyl,thienyl, quinolinyl, indolyl, and thiazolyl.

Alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, alkylamino, aryl, and heteroaryl mentioned aboveinclude both substituted and unsubstituted moieties. Possiblesubstituents on alkylamino, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, aryl, and heteroaryl include, but are not limitedto, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₃-C₂₀ cycloalkyl,C₃-C₂₀ cycloalkenyl, C₁-C₂₀ heterocycloalkyl, C₁-C₂₀ heterocycloalkenyl,C₁-C₁₀ alkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C₁-C₁₀alkylamino, arylamino, hydroxy, halo, oxo (0═), thioxo (S═), thio,silyl, C₁-C₁₀ alkylthio, arylthio, C₁-C₁₀ alkylsulfonyl, arylsulfonyl,acylamino, aminoacyl, aminothioacyl, amidino, mercapto, amido,thioureido, thiocyanato, sulfonamido, guanidine, ureido, cyano, nitro,acyl, thioacyl, acyloxy, carbamido, carbamyl, carboxyl, and carboxylicester. On the other hand, possible substituents on alkyl, alkenyl, oralkynyl include all of the above-recited substituents except C₁-C₁₀alkyl. Cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl,aryl, and heteroaryl can also be fused with each other.

“Amino” means a nitrogen moiety having two further substituents whereeach substituent has a hydrogen or carbon atom alpha bonded to thenitrogen. Unless indicated otherwise, the compounds of the inventioncontaining amino moieties may include protected derivatives thereof.Suitable protecting groups for amino moieties include acetyl,tert-butoxycarbonyl, benzyloxycarbonyl, and the like.

“Aromatic” means a moiety wherein the constituent atoms make up anunsaturated ring system, all atoms in the ring system are sp2 hybridizedand the total number of pi electrons is equal to 4n+2. An aromatic ringmay be such that the ring atoms are only carbon atoms or may includecarbon and non-carbon atoms (see Heteroaryl).

“Carbamoyl” means the radical —OC(O)NR_(a)R_(b) where R_(a) and R_(b)are each independently two further substituents where a hydrogen orcarbon atom is alpha to the nitrogen. It is noted that carbamoylmoieties may include protected derivatives thereof. Examples of suitableprotecting groups for carbamoyl moieties include acetyl,tert-butoxycarbonyl, benzyloxycarbonyl, and the like. It is noted thatboth the unprotected and protected derivatives fall within the scope ofthe invention.

“Carbonyl” means the radical —C(O)—. It is noted that the carbonylradical may be further substituted with a variety of substituents toform different carbonyl groups including acids, acid halides, amides,esters, and ketones.

“Carboxy” means the radical —C(O)O—. It is noted that compounds of theinvention containing carboxy moieties may include protected derivativesthereof, i.e. , where the oxygen is substituted with a protecting group.Suitable protecting groups for carboxy moieties include benzyl,tert-butyl, and the like.

“Cyano” means the radical —CN.

“Formyl” means the radical CH═O.

“Formimino” means the radical HC═NH.

“Halo” means fluoro, chloro, bromo or iodo.

“Halo-substituted alkyl”, as an isolated group or part of a largergroup, means “alkyl” substituted by one or more “halo” atoms, as suchterms are defined in this Application. Halo-substituted alkyl includeshaloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like.

“Hydroxy” means the radical —OH.

“Imine derivative” means a derivative comprising the moiety —C(═NR)—,wherein R comprises a hydrogen or carbon atom alpha to the nitrogen.

“Isomers” mean any compound having identical molecular formulae butdiffering in the nature or sequence of bonding of their atoms or in thearrangement of their atoms in space. Isomers that differ in thearrangement of their atoms in space are termed “stereoisomers.”Stereoisomers that are not mirror images of one another are termed“diastereomers” and stereoisomers that are nonsuperimposable mirrorimages are termed “enantiomers” or sometimes “optical isomers.” A carbonatom bonded to four nonidentical substituents is termed a “chiralcenter.” A compound with one chiral center has two enantiomeric forms ofopposite chirality. A mixture of the two enantiomeric forms is termed a“racemic mixture.”

“Nitro” means the radical —NO₂.

“Protected derivatives” means derivatives of compounds in which areactive site are blocked with protecting groups. Protected derivativesare useful in the preparation of pharmaceuticals or in themselves may beactive as inhibitors. A comprehensive list of suitable protecting groupscan be found in T.W.Greene, Protecting Groups in Organic Synthesis, 3rdedition, Wiley & Sons, 1999.

The term “substituted” means that an atom or group of atoms has replacedhydrogen as the substituent attached to another group. For aryl andheteroaryl groups, the term “substituted” refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.The term “unsubstituted” means that a given moiety may consist of onlyhydrogen substituents through available valencies (unsubstituted).

If a functional group is described as being “optionally substituted,”the function group may be either (1) not substituted, or (2)substituted. If a carbon of a functional group is described as beingoptionally substituted with one or more of a list of substituents, oneor more of the hydrogen atoms on the carbon (to the extent there areany) may separately and/or together be replaced with an independentlyselected optional substituent.

“Sulfide” means —S—R wherein R is H, alkyl, carbocycle, heterocycle,carbocycloalkyl or heterocycloalkyl. Particular sulfide groups aremercapto, alkylsulfide, for example methylsulfide (—S-Me); arylsulfide,e.g., phenylsulfide; aralkylsulfide, e.g., benzylsulfide.

“Sulfinyl” means the radical —S(O)—. It is noted that the sulfinylradical may be further substituted with a variety of substituents toform different sulfinyl groups including sulfinic acids, sulfinamides,sulfinyl esters, and sulfoxides.

“Sulfonyl” means the radical —S(O)(O)—. It is noted that the sulfonylradical may be further substituted with a variety of substituents toform different sulfonyl groups including sulfonic acids, sulfonamides,sulfonate esters, and sulfones.

“Thiocarbonyl” means the radical —C(S)—. It is noted that thethiocarbonyl radical may be further substituted with a variety ofsubstituents to form different thiocarbonyl groups including thioacids,thioamides, thioesters, and thioketones.

Certain of the compounds described herein may exist in variousstereoisomeric or tautomeric forms. Stereoisomers are compounds whichdiffer only in their spatial arrangement. When a disclosed compound isnamed or depicted by structure without indicating stereochemistry, it isunderstood that the name or structure encompasses all possiblestereoisomers, geometric isomers, including essentially pure stereo orgeometric isomers, as well as combination thereof.

It is to be understood that when a compound herein is represented by astructural formula or designated by a chemical name herein, all othertautomeric forms which may exist for the compound are encompassed by thestructural formula.

Certain of the disclosed compounds may exist in various stereoisomericforms. Stereoisomers are compounds that differ only in their spatialarrangement. Enantiomers are pairs of stereoisomers whose mirror imagesare not superimposable, most commonly because they contain anasymmetrically substituted carbon atom that acts as a chiral center.“Enantiomer” means one of a pair of molecules that are mirror images ofeach other and are not superimposable. Diastereomers are stereoisomersthat contain two or more asymmetrically substituted carbon atoms.

When the stereochemistry of a disclosed compound is named or depicted bystructure, the named or depicted stereoisomer is at least 60%, 70%, 80%,90%, 99% or 99.9% by weight pure relative to all of the otherstereoisomers. Percent by weight pure relative to all of the otherstereoisomers is the ratio of the weight of one stereoisomer over theweight of the other stereoisomers. When a single enantiomer is named ordepicted by structure, the depicted or named enantiomer is at least 60%,70%, 80%, 90%, 99% or 99.9% by weight optically pure (also referred toas “enantiomerically pure”). Percent optical purity by weight is theratio of the weight of the enantiomer over the weight of the enantiomerplus the weight of its optical isomer.

When the stereochemistry of a disclosed compound is named or depicted bystructure, and the named or depicted structure encompasses more than onestereoisomer (e.g., as in a diastereomeric pair), it is to be understoodthat one of the encompassed stereoisomers or any mixture of theencompassed stereoisomers are included. It is to be further understoodthat the stereoisomeric purity of the named or depicted stereoisomers atleast 60%, 70%, 80%, 90%, 99% or 99.9% by weight pure relative to all ofthe other stereoisomers. The stereoisomeric purity in this case isdetermined by dividing the total weight in the mixture of thestereoisomers encompassed by the name or structure by the total weightin the mixture of all of the stereoisomers.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry, and the compound has one chiral center,it is to be understood that the name or structure encompasses oneenantiomer of compound free from the corresponding optical isomer, aracemic mixture of the compound and mixtures enriched in one enantiomerrelative to its corresponding optical isomer.

When a disclosed compound is named or depicted by structure withoutindicating the stereochemistry and e.g., the compound has at least twochiral centers, it is to be understood that the name or structureencompasses one stereoisomer free of other stereoisomers, mixtures ofstereoisomers, and mixtures of stereoisomers in which one or morestereoisomers is enriched relative to the other stereoisomer(s). Forexample, the name or structure may encompass one stereoisomer free ofother diastereomers, mixtures of stereoisomers, and mixtures ofstereoisomers in which one or more diastereomers is enriched relative tothe other diastereomer(s).

Enantiomeric and diastereomeric mixtures can be resolved into theircomponent enantiomers or stereoisomers by well-known methods, such aschiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers anddiastereomers can also be obtained from diastereomerically- orenantiomerically-pure intermediates, reagents, and catalysts bywell-known asymmetric synthetic methods.

“Animal” includes humans, non-human mammals (e.g., non-human primates,rodents, mice, rats, hamsters, dogs, cats, rabbits, cattle, horses,sheep, goats, swine, deer, and the like) and non-mammals (e.g., birds,and the like).

“Bioavailability” as used herein is the fraction or percentage of anadministered dose of a drug or pharmaceutical composition that reachesthe systemic circulation intact. In general, when a medication isadministered intravenously, its bioavailability is 100%. However, when amedication is administered via other routes (e.g., orally), itsbioavailability decreases (e.g., due to incomplete absorption andfirst-pass metabolism). Methods to improve the bioavailability includeprodrug approach, salt synthesis, particle size reduction, complexation,change in physical form, solid dispersions, spray drying, and hot-meltextrusion.

“Disease” specifically includes any unhealthy condition of an animal orpart thereof and includes an unhealthy condition that may be caused by,or incident to, medical or veterinary therapy applied to that animal,i.e., the “side effects” of such therapy.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means organic or inorganic salts ofcompounds of the present invention which are pharmaceuticallyacceptable, as defined above, and which possess the desiredpharmacological activity. Such salts include acid addition salts formedwith inorganic acids, or with organic acids. Pharmaceutically acceptablesalts also include base addition salts which may be formed when acidicprotons present are capable of reacting with inorganic or organic bases.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate “mesylate,”ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g.,sodium and potassium) salts, alkaline earth metal (e.g., magnesium)salts, and ammonium salts. A pharmaceutically acceptable salt mayinvolve the inclusion of another molecule such as an acetate ion, asuccinate ion or other counter ion. The counter ion may be any organicor inorganic moiety that stabilizes the charge on the parent compound.Furthermore, a pharmaceutically acceptable salt may have more than onecharged atom in its structure. Instances where multiple charged atomsare part of the pharmaceutically acceptable salt can have multiplecounter ions. Hence, a pharmaceutically acceptable salt can have one ormore charged atoms and/or one or more counter ion.

“Pharmaceutically acceptable carrier” means a non-toxic solvent,dispersant, excipient, adjuvant, or other material which is mixed withthe compounds of the present invention in order to form a pharmaceuticalcomposition, i.e., a dose form capable of administration to the patient.Examples of pharmaceutically acceptable carrier includes suitablepolyethylene glycol (e.g., PEG400), surfactant (e.g., Cremophor), orcyclopolysaccharide (e.g., hydroxypropyl-β-cyclodextrin or sulfobutylether β-cyclodextrins), polymer, liposome, micelle, nanosphere, etc.

“Pharmacophore,” as defined by The International Union of Pure andApplied Chemistry, is an ensemble of steric and electronic features thatis necessary to ensure the optimal supramolecular interactions with aspecific biological target and to trigger (or block) its biologicalresponse. For example, Camptothecin is the pharmacophore of the wellknown drug topotecan and irinotecan. Mechlorethamine is thepharmacophore of a list of widely used nitrogen mustard drugs likeMelphalan, Cyclophosphamide, Bendamustine, and so on.

“Prodrug” means a compound that is convertible in vivo metabolicallyinto an active pharmaceutical according to the present invention. Forexample, an inhibitor comprising a hydroxyl group may be administered asan ester that is converted by hydrolysis in vivo to the hydroxylcompound.

“Stability” in general refers to the length of time a drug retains itsproperties without loss of potency. Sometimes this is referred to asshelf life. Factors affecting drug stability include, among otherthings, the chemical structure of the drug, impurity in the formulation,pH, moisture content, as well as environmental factors such astemperature, oxidization, light, and relative humidity. Stability can beimproved by providing suitable chemical and/or crystal modifications(e.g., surface modifications that can change hydration kinetics;different crystals that can have different properties), excipients(e.g., anything other than the active substance in the dosage form),packaging conditions, storage conditions, etc.

“Therapeutically effective amount” of a composition described herein ismeant an amount of the composition which confers a therapeutic effect onthe treated subject, at a reasonable benefit/risk ratio applicable toany medical treatment. The therapeutic effect may be objective (i.e.,measurable by some test or marker) or subjective (i.e., subject gives anindication of or feels an effect). An effective amount of thecomposition described above may range from about 0.1 mg/kg to about 500mg/kg, preferably from about 0.2 to about 50 mg/kg. Effective doses willalso vary depending on route of administration, as well as thepossibility of co-usage with other agents. It will be understood,however, that the total daily usage of the compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;the activity of the specific compound employed; the specific compositionemployed; the age, body weight, general health, sex and diet of thepatient; the time of administration, route of administration, and rateof excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or contemporaneously with thespecific compound employed; and like factors well known in the medicalarts.

As used herein, the term “treating” refers to administering a compoundto a subject that has a neoplastic or immune disorder, or has a symptomof or a predisposition toward it, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisorder, the symptoms of or the predisposition toward the disorder. Theterm “an effective amount” refers to the amount of the active agent thatis required to confer the intended therapeutic effect in the subject.Effective amounts may vary, as recognized by those skilled in the art,depending on route of administration, excipient usage, and thepossibility of co-usage with other agents.

A “subject” refers to a human and a non-human animal. Examples of anon-human animal include all vertebrates, e.g., mammals, such asnon-human primates (particularly higher primates), dog, rodent (e.g.,mouse or rat), guinea pig, cat, and non-mammals, such as birds,amphibians, reptiles, etc. In a preferred embodiment, the subject is ahuman. In another embodiment, the subject is an experimental animal oranimal suitable as a disease model.

“Combination therapy” includes the administration of the subjectcompounds of the present invention in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different antineoplastic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment). For instance, thecompounds of the invention can be used in combination with otherpharmaceutically active compounds, or non-drug therapies, preferablycompounds that are able to enhance the effect of the compounds of theinvention. The compounds of the invention can be administeredsimultaneously (as a single preparation or separate preparation) orsequentially to the other therapies. In general, a combination therapyenvisions administration of two or more drugs/treatments during a singlecycle or course of therapy.

In one embodiment, the compounds of the invention are administered incombination with one or more of traditional chemotherapeutic agents. Thetraditional chemotherapeutic agents encompass a wide range oftherapeutic treatments in the field of oncology. These agents areadministered at various stages of the disease for the purposes ofshrinking tumors, destroying remaining cancer cells left over aftersurgery, inducing remission, maintaining remission and/or alleviatingsymptoms relating to the cancer or its treatment. Examples of suchagents include, but are not limited to, alkylating agents such asNitrogen Mustards (e.g., Bendamustine, Cyclophosphamide, Melphalan,Chlorambucil, Isofosfamide), Nitrosureas (e.g., Carmustine, Lomustineand Streptozocin), ethylenimines (e.g., thiotepa, hexamethylmelanine),Alkylsulfonates (e.g., Busulfan), Hydrazines and Triazines (e.g.,Altretamine, Procarbazine, Dacarbazine and Temozolomide), and platinumbased agents (e.g., Carboplatin, Cisplatin, and Oxaliplatin); plantalkaloids such as Podophyllotoxins (e.g., Etoposide and Tenisopide),Taxanes (e.g., Paclitaxel and Docetaxel), Vinca alkaloids (e.g.,Vincristine, Vinblastine and Vinorelbine); anti-tumor antibiotics suchas Chromomycins (e.g., Dactinomycin and Plicamycin), Anthracyclines(e.g., Doxorubicin, Daunorubicin, Epirubicin, Mitoxantrone, andIdarubicin), and miscellaneous antibiotics such as Mitomycin andBleomycin; anti-metabolites such as folic acid antagonists (e.g.,Methotrexate), pyrimidine antagonists (e.g., 5-Fluorouracil, Foxuridine,Cytarabine, Capecitabine, and Gemcitabine), purine antagonists (e.g.,6-Mercaptopurine and 6-Thioguanine) and adenosine deaminase inhibitors(e.g., Cladribine, Fludarabine, Nelarabine and Pentostatin);topoisomerase inhibitors such as topoisomerase I inhibitors(Topotecan,Irinotecan), topoisomerase II inhibitors (e.g., Amsacrine, Etoposide,Etoposide phosphate, Teniposide), and miscellaneous anti-neoplasticssuch as ribonucleotide reductase inhibitors (Hydroxyurea),adrenocortical steroid inhibitor (Mitotane), anti-microtubule agents(Estramustine), and retinoids (Bexarotene, Isotretinoin, Tretinoin(ATRA).

In one aspect of the invention, the compounds may be administered incombination with one or more targeted anti-cancer agents that modulateprotein kinases involved in various disease states. Examples of suchkinases may include, but are not limited ABL1, ABL2/ARG, ACK1, AKT1,AKT2, AKT3, ALK, ALK1/ACVRL1, ALK2/ACVR1, ALK4/ACVR1B, ALK5/TGFBR1,ALK6/BMPR1B, AMPK(A1/B1/G1), AMPK(A1/B1/G2), AMPK(A1/B1/G3),AMPK(A1/B2/G1), AMPK(A2/B1/G1), AMPK(A2/B2/G1), AMPK(A2/B2/G2), ARAF,ARK5/NUAK1, ASK1/MAP3K5, ATM, Aurora A, Aurora B , Aurora C , AXL, BLK,BMPR2, BMX/ETK, BRAF, BRK, BRSK1, BRSK2, BTK, CAMK1a , CAMK1b, CAMK1d,CAMK1g , CAMKIIa , CAMKIIb , CAMKIId , CAMKIIg , CAMK4, CAMKK1, CAMKK2,CDC7-DBF4, CDK1-cyclin A, CDK1-cyclin B, CDK1-cyclin E, CDK2-cyclin A,CDK2-cyclin A1, CDK2-cyclin E, CDK3-cyclin E, CDK4-cyclin D1,CDK4-cyclin D3, CDK5-p25, CDK5-p35, CDK6-cyclin D1, CDK6-cyclin D3,CDK7-cyclin H, CDK9-cyclin K, CDK9-cyclin Tl, CHK1, CHK2, CK1a1, CK1d ,CK1epsilon , CK1g1 , CK1g2, CK1g3 , CK2a , CK2a2, c-KIT, CLK1 , CLK2,CLK3, CLK4, c-MER, c-MET, COT1/MAP3K8, CSK, c-SRC, CSF1R, CTK/MATK,DAPK1, DAPK2, DCAMKL1, DCAMKL2, DDR1, DDR2, DLK/MAP3K12, DMPK,DMPK2/CDC42BPG, DNA-PK, DRAK1/STK17A, DYRK1/DYRK1A, DYRK1B, DYRK2,DYRK3, DYRK4, EEF2K, EGFR, EIF2AK1, EIF2AK2, EIF2AK3, EIF2AK4/GCN2,EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2,EPHB3, EPHB4, ERBB2/HER2, ERBB4/HER4, ERK1/MAPK3, ERK2/MAPK1,ERK5/MAPK7, FAK/PTK2, FER, FES/FPS, FGFR1, FGFR2, FGFR3, FGFR4, FGR,FLT1/VEGFR1, FLT3, FLT4/VEGFR3, FMS, FRK/PTK5, FYN, GCK/MAP4K2, GRK1,GRK2, GRK3, GRK4, GRK5, GRK6, GRK7, GSK3a, GSK3b, Haspin, HCK,HGK/MAP4K4, HIPK1, HIPK2, HIPK3, HIPK4, HPK1/MAP4K1, IGF1R, IKKa/CHUK ,IKKb/IKBKB, IKKe/IKBKE, IR, IRAK1, IRAK4, IRR/INSRR, ITK, JAK1, JAK2,JAK3, JNK1 , JNK2 , JNK3, KDR/VEG1-R2, KHS/MAP4K5, LATS1, LATS2, LCK,LCK2/ICK, LKB1 , LIMK1, LOK/STK10, LRRK2, LYN, LYNB, MAPKAPK2, MAPKAPK3,MAPKAPK5/PRAK, MARK1, MARK2/PAR-1Ba, MARK3, MARK4, MEK1, MEK2, MEKK1,MEKK2, MEKK3, MELK, MINK/MINK1, MKK4, MKK6, MLCK/MYLK, MLCK2/MYLK2,MLK1/MAP3K9, MLK2/MAP3K10, MLK3/MAP3K11, MNK1, MNK2, MRCKa/, CDC42BPA,MRCKb/, CDC42BPB, MSK1/RPS6KA5, MSK2/RPS6KA4, MSSK1/STK23, MST1/STK4,MST2/STK3, MST3/STK24, MST4, mTOR/FRAP1, MUSK, MYLK3, MYO3b, NEK1, NEK2,NEK3, NEK4, NEK6, NEK7, NEK9, NEK11, NIK/MAP3K14, NLK, OSR1/OXSR1,P38a/MAPK14, P38b/MAPK11, P38d/MAPK13 , P38g/MAPK12 , P70S6K/RPS6KB1,p70S6Kb/, RPS6KB2, PAK1, PAK2, PAK3, PAK4, PAK5, PAK6, PASK, PBK/TOPK,PDGFRa, PDGFRb, PDK1/PDPK1, PDK1/PDHK1, PDK2/PDHK2 , PDK3/PDHK3,PDK4/PDHK4, PHKg1 , PHKg2 , PI3Ka, (p110a/p85a), PI3Kb, (p110b/p85a),PI3Kd, (p110d/p85a), PI3Kg(p120g), PIM1, PIM2, PIM3, PKA, PKAcb, PKAcg ,PKCa , PKCb1 , PKCb2 , PKCd , PKCepsilon, PKCeta, PKCg , PKCiota,PKCmu/PRKD1, PKCnu/PRKD3, PKCtheta, PKCzeta, PKD2/PRKD2, PKG1a , PKG1b ,PKG2/PRKG2, PKN1/PRK1, PKN2/PRK2, PKN3/PRK3, PLK1, PLK2, PLK3, PLK4/SAK,PRKX, PYK2, RAF1, RET, RIPK2, RIPK3, RIPK5, ROCK1, ROCK2, RON/MST1R,ROS/ROS1, RSK1, RSK2, RSK3, RSK4, SGK1, SGK2, SGK3/SGKL, SIK1, SIK2,SLK/STK2, SNARK/NUAK2, SRMS, SSTK/TSSK6, STK16, STK22D/TSSK1,STK25/YSK1, STK32b/YANK2, STK32c/YANK3, STK33, STK38/NDR1, STK38L/NDR2,STK39/STLK3, SRPK1, SRPK2, SYK, TAK1, TAOK1, TAOK2/TAO1, TAOK3/JIK,TBK1, TEC, TESK1, TGFBR2, TIE2/TEK, TLK1, TLK2, TNIK, TNK1, TRKA, TRKB,TRKC, TRPM7/CHAK1, TSSK2, TSSK3/STK22C, TTBK1, TTBK2, TTK, TXK,TYK1/LTK, TYK2, TYRO3/SKY, ULK1, ULK2, ULK3, VRK1, VRK2, WEE1, WNK1,WNK2, WNK3, YES/YES1, ZAK/MLTK, ZAP70, ZIPK/DAPK3, KINASE, MUTANTS,ABL1(E255K), ABL1(F317I), ABL1(G250E), ABL1(H396P), ABL1(M351T),ABL1(Q252H), ABL1(T315I), ABL1(Y253F), ALK (C1156Y), ALK(L1196M), ALK(F1174L), ALK (R1275Q), BRAF(V599E), BTK(E41K), CHK2(I157T),c-Kit(A829P), c-KIT(D816H), c-KIT(D816V), c-Kit(D820E), c-Kit(N822K),C-Kit (T670I), c-Kit(V559D), c-Kit(V559D/V654A), c-Kit(V559D/T670I),C-Kit (V560G), c-KIT(V654A), C-MET(D1228H), C-MET(D1228N),C-MET(F1200I), c-MET(M1250T), C-MET(Y1230A), C-MET(Y1230C),C-MET(Y1230D), C-MET(Y1230H), c-Src(T341M), EGFR(G719C), EGFR(G719S),EGFR(L858R), EGFR(L861Q), EGFR(T790M), EGFR, (L858R,T790M) ,EGFR(d746-750/T790M), EGFR(d746-750), EGFR(d747-749/A750P),EGFR(d747-752/P753S), EGFR(d752-759), FGFR1(V561M), FGFR2(N549H),FGFR3(G697C), FGFR3(K650E), FGFR3(K650M), FGFR4(N535K), FGFR4(V550E),FGFR4(V550L), FLT3(D835Y), FLT3(ITD), JAK2 (V617F), LRRK2 (G2019S),LRRK2 (I2020T), LRRK2 (R1441C), p38a(T106M), PDGFRa(D842V),PDGFRa(T674I), PDGFRa(V561D), RET(E762Q), RET(G691S), RET(M918T),RET(R749T), RET(R813Q), RET(V804L), RET(V804M), RET(Y791F), TIF2(R849W),TIF2(Y897S), and TIF2(Y1108F).

In another aspect of the invention, the subject compounds may beadministered in combination with one or more targeted anti-cancer agentsthat modulate non-kinase biological targets, pathway, or processes. Suchtargets pathways, or processes include but not limited to heat shockproteins (e.g. HSP90), poly-ADP (adenosine diphosphate)-ribosepolymerase (PARP), hypoxia-inducible factors(HIF), proteasome,Wnt/Hedgehog/Notch signaling proteins, TNF-alpha, matrixmetalloproteinase, farnesyl transferase, apoptosis pathway (e.g Bcl-xL,Bcl-2, Bcl-w), histone deacetylases (HDAC), histone acetyltransferases(HAT), and methyltransferase (e.g histone lysine methyltransferases,histone arginine methyltransferase, DNA methyltransferase, etc), andother immunotherapies(e.g anti-PD1, anti-PDL1, anti-CTLA4, A2Aantagonist etc).

In another aspect of the invention, the compounds of the invention areadministered in combination with one or more of other anti-cancer agentsthat include, but are not limited to, gene therapy, RNAi cancer therapy,chemoprotective agents (e.g., amfostine, mesna, and dexrazoxane),antibody conjugate(e.g brentuximab vedotin, ibritumomab tioxetan),cancer immunotherapy such as Interleukin-2, cancer vaccines(e.g.,sipuleucel-T) or monoclonal antibodies (e.g., Bevacizumab, Alemtuzumab,Rituximab, Trastuzumab, etc).

In another aspect of the invention, the subject compounds areadministered in combination with radiation therapy or surgeries.Radiation is commonly delivered internally (implantation of radioactivematerial near cancer site) or externally from a machine that employsphoton (x-ray or gamma-ray) or particle radiation. Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In certain embodiments, the compounds of the invention are administeredin combination with one or more of radiation therapy, surgery, oranti-cancer agents that include, but are not limited to, DNA damagingagents, anti-metabolites, topoisomerase inhibitors, anti-microtubuleagents, kinase inhibitors, epigenetic agents, HSP90 inhibitors, PARPinhibitors, and antibodies targeting VEGF, HER2, EGFR, CD50, CD20, CD30,CD33, etc.

In certain embodiments, the compounds of the invention are administeredin combination with one or more of abarelix, abiraterone acetate,aldesleukin, alemtuzumab, altretamine, anastrozole, asparaginase,bendamustine, bevacizumab, bexarotene, bicalutamide, bleomycin,bortezombi, brentuximab vedotin, busulfan, capecitabine, carboplatin,carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine,clomifene, crizotinib, cyclophosphamide, dasatinib, daunorubicinliposomal, decitabine, degarelix, denileukin diftitox, denileukindiftitox, denosumab, docetaxel, doxorubicin, doxorubicin liposomal,epirubicin, eribulin mesylate, erlotinib, estramustine, etoposidephosphate, everolimus, exemestane, fludarabine, fluorouracil,fotemustine, fulvestrant, gefitinib, gemcitabine, gemtuzumab ozogamicin,goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab tiuxetan,idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a,ipilimumab, ixabepilone, lapatinib ditosylate, lenalidomide, letrozole,leucovorin, leuprolide acetate, levamisole, lomustine, mechlorethamine,melphalan, methotrexate, mitomycin C, mitoxantrone, nelarabine,nilotinib, oxaliplatin, paclitaxel, paclitaxel protein-bound particle,pamidronate, panitumumab, pegaspargase, peginterferon alfa-2b,pemetrexed disodium, pentostatin, raloxifene, rituximab, sorafenib,streptozocin, sunitinib maleate, tamoxifen, temsirolimus, teniposide,thalidomide, toremifene, tositumomab, trastuzumab, tretinoin,uramustine, vandetanib, vemurafenib, vinorelbine, zoledronate, radiationtherapy, or surgery.

The invention further provides methods for the prevention or treatmentof a neoplastic disease or autoimmune disease. In one embodiment, theinvention relates to a method of treating a neoplastic disease orautoimmune disease, in a subject in need of treatment comprisingadministering to said subject a therapeutically effective amount of acompound of the invention. In one embodiment, the invention furtherprovides for the use of a compound of the invention in the manufactureof a medicament for halting or decreasing a neoplastic disease orautoimmune disease.

In certain embodiments, the neoplastic disease is a lung cancer, headand neck cancer, central nervous system cancer, prostate cancer,testicular cancer, colorectal cancer, pancreatic cancer, liver cancer,stomach cancer, biliary tract cancer, esophageal cancer,gastrointestinal stromal tumor, breast cancer, cervical cancer, ovariancancer, uterine cancer, leukemia, lymphomas, multiple myeloma, melanoma,basal cell carcinoma, squamous cell carcinoma, bladder cancer, renalcancer, sarcoma, mesothelioma, thymoma, myelodysplastic syndrome, ormyeloproliferative disease.

The autoimmune diseases that can be affected using compounds andcompositions according to the invention include, but are not limited toallergy, Alzheimer's disease, acute disseminated encephalomyelitis,Addison's disease, ankylosing spondylitis, antiphospholipid antibodysyndrome, asthma, atherosclerosis, autoimmune hemolytic anemia,autoimmune hemolytic and thrombocytopenic states, autoimmune hepatitis,autoimmune inner ear disease, bullous pemphigoid, coeliac disease,chagas disease, chronic obstructive pulmonary disease, chronicIdiopathic thrombocytopenic purpura (ITP), churg-strauss syndrome,Crohn's disease, dermatomyositis, diabetes mellitus type 1,endometriosis, Goodpasture's syndrome (and associated glomerulonephritisand pulmonary hemorrhage), graves' disease, guillain-bane syndrome,hashimoto's disease, hidradenitis suppurativa, idiopathicthrombocytopenic purpura, interstitial cystitis, irritable bowelsyndrome, lupus erythematosus, morphea, multiple sclerosis, myastheniagravis, narcolepsy, neuromyotonia, Parkinson's disease, pemphigusvulgaris, pernicious anaemia, polymyositis, primary biliary cirrhosis,psoriasis, psoriatic arthritis, rheumatoid arthritis, schizophrenia,septic shock, scleroderma, Sjogren's disease, systemic lupuserythematosus (and associated glomerulonephritis), temporal arteritis,tissue graft rejection and hyperacute rejection of transplanted organs,vasculitis (ANCA-associated and other vasculitides), vitiligo, andwegener's granulomatosis.

It should be understood that the invention is not limited to theparticular embodiments shown and described herein, but that variouschanges and modifications may be made without departing from the spiritand scope of the invention as defined by the claims.

The compounds according to the present invention may be synthesizedaccording to a variety of schemes. Necessary starting materials may beobtained by standard procedures of organic chemistry. The compounds andprocesses of the present invention will be better understood inconnection with the following representative synthetic schemes andexamples, which are intended as an illustration only and not limiting ofthe scope of the invention. Various changes and modifications to thedisclosed embodiments will be apparent to those skilled in the art andsuch changes and modifications including, without limitation, thoserelating to the chemical structures, substituents, derivatives, and/ormethods of the invention may be made without departing from the spiritof the invention and the scope of the appended claims.

A typical approach to synthesize of the intermediate

in which W is N is described in Scheme 1. The intermedediate with N isCH can be made similarly.

In Scheme 1, the starting material 1-1 is protected with SEM group togive intermediate 1-2, which is followed by Buckwald coupling to yieldintermediate 1-3. Hydrolysis of imine 1-3 gives aniline intermediate1-4. Condensation of 1-4 with acetic acid anhydride results inSEM-protected tricyclic intermediate 1-5.

A typical approach to synthesize of the intermediate

is described in Scheme 2:

In Scheme 2, the appropriate ketone starting material 2-1 can react withtribromophosphine to form the aldehyde intermediate 2-2, which cancouple with Boc-protected piperazine to form the intermediate 2-3. Afterthat, 2-3 will couple with appropriate phenylboronic acid via a Suzukireaction to form intermediate 2-4, followed by a de-boc process to yieldkey intermediate 2-5.

A typical approach to synthesize of the intermediate

is described in Scheme 3:

In Scheme 3, the staring intermediate 1-5 reacts with ethyl4-Br-2-F-benzoic ester under basic conditions to give 3-2. Buckwaldcoupling or nucleophilic substitution reactions between 3-2 and 5-5produces intermediate 3-3. Hydrolysis of 3-3 gives intermediate 3-4.

A typical approach to synthesize of the intermediate

in which R₅ is NO₂ is described in Scheme 4:

In Scheme 4, the staring intermediate 4-1 reacts with appropriatealcohol or amine will yield 4-2.

A typical approach to synthesize of the Formula (I) compounds with Z₁=NHor 0 is described in Scheme A:

The intermediate 3-4 can undergo an amide coupling with ammonia givesintermediate A-2. Sequential substitution reaction of chlorobenzenesulfonyl halides (X is Cl or F) with appropriate amine NH₂LR₄ or alcoholHO-L-R₄ and coupling reaction with intermediate A-2 followed bydeprotection of SEM group leads to Formula (I).

Similarly, a typical approach to synthesize of the Forumula (I)compounds is described in Scheme B:

In Scheme B, The intermediate 3-4 can undergo an amide coupling withammonia gives intermediate A-2. After that, the substitution reaction ofappropriate sulfonyl chloride followed by deprotection of SEM groupleads to final product Formula (I).

Similarly, a typical approach to synthesize of the Forumula (I)compounds is described in Scheme C:

In Scheme C, the amide coupling of 3-4 and 4-2 leads to final productFormula (I).

The compounds and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the chemicalstructures, substituents, derivatives, formulations and/or methods ofthe invention may be made without departing from the spirit of theinvention and the scope of the appended claims.

Where NMR data are presented, ¹H spectra were obtained on XL400 (400MHz) and are reported as ppm down field from Me₄Si with number ofprotons, multiplicities, and coupling constants in Hertz indicatedparenthetically. Where HPLC data are presented, analyses were performedusing an Agilent 1100 system. Where LC/MS data are presented, analyseswere performed using an Applied Biosystems API-100 mass spectrometer andShimadzu SCL-10A LC column:

EXAMPLE 1 Preparation of Key Intermediate IM-14-1

Synthesis of 2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde 2: Asolution of anhydrous chloroform (57 ml) and anhydrousN,N-dimethylformamide (9 mL) was cooled to ˜3° C. (internal temperature)under nitrogen before phosphorus tribromide (10 mL, 0.1 mol) wasintroduced dropwise at a rate so that the reaction was maintained at ˜3°C. After the addition was complete the reaction was allowed to warmslowly to ˜10° C. and then the temperature was raised to 70° C. where itwas maintained for 30 min. The reaction was cooled to rt and3,3-dimethylcyclohexanone 1 (5 g, 0.04 mol) was added slowly over 20min. After the addition was complete the reaction was warmed to 70° C.and it was stirred for 1.5 h. The mixture was then cooled to 0° C. and asolution of 4M sodium acetate (53 ml) was added slowly. The pH of theresulting solution was adjusted to ˜7 using a solution of 5M NaOH andthe mixture was then extracted with heptanes (100 mL×3). The combinedorganic fractions were dried (Na₂SO₄), filtered and concentrated underreduced pressure to give 2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde2 (4 g, 49%)as a yellow oil.

Synthesis of 2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde3: To a degassed solution of2-bromo-4,4-dimethylcyclohex-1-enecarbaldehyde 2 (5 g, 0.023 mol) and4-chlorophenyl boronic acid (3.6 g, 0.023 mol) in 1,4-dioxane (50 mL) atrt was added a solution of 2M Na₂CO₃ (20.4 ml). Nitrogen was bubbledthrough the mixture for 2 min and then PdCl₂(dppf) (0.5 g) was added.The reaction flask was heated to 120° C. where it was maintained for 3h. After this time the suspension was cooled to rt and filtered throughCelite. The collected solids were washed with additional dichloromethaneand the combined filtrate and washings were concentrated under reducedpressure. Purification by column chromatography on silica with PE:EA=20:1 gave 2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde 3(3 g, 53%) as a white solid. MS: 249[M+H]⁺

Synthesis of (2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4:A solution of 2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enecarbaldehyde3 (20 g, 80.6 mmol) in MeOH (100 mL) was cooled to 0° C., NaBH₄ (3.1g,80.6 mmol) was added portionwise to the reaction at a rate so that thereaction was maintained at 0˜5° C. After added, the mixture was stirredfor 1 h at 0° C. Water was added slowly to the mixture and extractedwith EA (200 mL×3), the organic layer was washed with brine and driedNa₂SO₄, filtered and concentrated under reduced pressure to give(2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4 (15 g, 75%)as a white solid. MS: 233[M+H−H₂O]⁺

Synthesis of1-(2-(bromomethyl)-5,5-dimethylcyclohex-1-enyl)-4-chlorobenzene 5: Asolution of (2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methanol 4(15 g, 0.060 mol) and in Et₂O (300 ml) was cooled to 0° C. beforephosphorus tribromide (7.5 mL) was added dropwise to the mixture, afteradded, the mixture was stirred for 1 h at 0° C. for 90 minutes. Thereaction mixture was added H₂O before being extracted with EA. Theorganic layer was washed with a saturated NaHCO₃ solution and brine anddried Na₂SO₄, filtered and concentrated under reduced pressure to give1-(2-(bromomethyl)-5,5-dimethylcyclohex-1-enyl)-4-chlorobenzene 5 (18 g,96%) as a colorless oil.

Synthesis of tert-butyl4-4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-carboxylate—Toa solution of 1-bromo-2-(bromomethyl)-5,5-dimethylcyclohex-1-ene 5 (21g, 0.067 mol) and tert-butyl piperazine-1-carboxylate (12.4 g, 0.067mol) in dichloromethane (200 ml) at rt was added TEA (12.2 g, 0.12 mol).The reaction was stirred for 2 h. The reaction mixture was concentratedunder reduced pressure to give the crude product. Purification by columnchromatography on silica with PE:EA=20:1 provided tert-butyl4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-carboxylate6 (21 g, 75%).

Synthesis of1-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazinehydrogen chloride: To a solution of tert-butyl4-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enyl)methyl)piperazine-1-carboxylate6 (30 g, 0.072 mol) in MeOH (20 ml) was added conc. HCl (50 mL). Thereaction was stirred for 24 hours and then concentrated under reducedpressure. A saturated solution of Na₂CO₃ was added to adjust the pH to˜8-9 and the mixture was extracted with dichloromethane (x2). Thecombined extracts were washed with brine, dried (Na₂SO₄), filtered andconcentrated under reduced pressure. The oil product was treated withMeOH/HCl(g) (3M, 500 mL) and stirred for 1 hour, then concentrated underreduced pressure to get the product1-((2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-enylnnethyl)piperazinehydrogen chloride IM-14-1 (23 g, 83%). MS: 319[M+H]⁺ ¹H NMR (400 MHz,DMSO) δ 11.51 (s, 1H), 9.60 (s, 1H), 9.18 (s, 1H), 7.45 (d, J=8.2 Hz,2H), 7.15 (d, J=8.0 Hz, 2H), 3.43 (s, 8H), 2.84 (s, 2H), 2.39 (s, 2H),2.03 (s, 2H), 1.45 (t, J=6.0 Hz, 2H), 0.96 (s, 6H).

EXAMPLE 2 Preparation of Key Intermediate IM-14-3

Step 1: To a solution of compound 1 (200 g, 1.3 mol) in a solution ofsulfuric acid (314 mL) and water (2.5 L) was added potassium iodide (217g, 1.3 mol) and KIO₃ (140 g, 655 mmol) at 20° C. The mixture was stirredat 80° C. for 4 h. TLC (petroleum ether:ethyl acetate=1:1, R_(f)=0.43)showed reactant was consumed completely, and one major new spot withlarger polarity was detected. The five reactions were combined forworkup. The mixture was cooled to RT and adjusted to pH 9 with thecareful addition of NaOH (5N). Sodium sulfite saturated solution wasadded with vigorous stirring until the iodine color had beendisappeared. A significant amount of brown solid remained out ofsolution, which was collected by filtration, washed with water (2000 mL)to give compound 2 (1.5 kg, 5.4 mol, 82.1% yield) as a brown solid. ¹HNMR: (400 MHz, DMSO-d₆) δ 8.50 (s, 1H), 2.58 (s, 3H).

Step 2: To a solution of compound 2 (387 g, 1.4 mol) in THF (2.4 L) wasadded TEA (422 g, 4.2 mol) and CuI (26.47 g, 139 mmol). The mixture wasdegassed and purged with nitrogen atmosphere for 3 times, Pd(PPh₃)₂Cl₂(29.3 g, 42 mmol) was added and the mixture was degassed and purged withnitrogen atmosphere for 3 times again. Then ethynyl(trimethyl)silane(164 g, 1.7 mol) was added drop-wise. During which the temperature wasmaintained below 30° C. The mixture was stirred at 25° C. for 3 h undernitrogen atmosphere. TLC (petroleum ether:ethyl acetate=1:1, R_(f)=0.43)show reactant was consumed completely, and one major new spot withlarger polarity was detected. The four reactions were combined forworkup. The mixture was filtered and concentrated under reduced pressureto give a residue. The resulting residue was dissolved in water (10 L)and extracted with ethyl acetate (4×1 L). The organic layer was washedwith water and then separated out. The organic layer dried overanhydrous sodium sulfate, filtered and concentrated under reducedpressure at 50° C. The solid was added to petroleum ether (300 mL) andstirred for 30 min Then the suspension was filtered and the solid wasdried in air to give compound 3 (1.3 kg, crude) as a brown solid. Thesolid was added to the solution of DCM (5 L) and stirred at 25° C. for 3h. Then filtered and concentrated to give compound 3 (700 g) as yellowsolid. ¹H NMR: (400 MHz, DMSO-d₆) δ 8.15 (s, 1H), 2.63 (s, 3H), 0.24 (s,9H).

Step 3: To a solution of t-BuOK (126 g, 1.1 mol, 2.0 eq) in NMP (500 mL)was added a solution of compound 3 (140 g, 561 mmol, 1.0 eq) in NMP (400mL) in dropwise at 140° C. The mixture was stirred at 140° C. for 3 h.LCMS showed the reaction was completed. The mixture was cooled to 20° C.and combined poured into saturated NaCl solution (5 L) and extractedwith ethyl acetate (5 L*3). The combined organic phase was washed withbrine (3 L), dried with anhydrous Na₂SO₄, filtered and concentrated invacuum to give compound 4 (140 g, 790.3 mmol, 35.2% yield) as yellowsolid. ¹H NMR: (400 MHz, DMSO-d₆) δ 12.20 (s, 1H), 8.68 (s, 1H), 7.61(t, J=2.1 Hz, 1H), 6.59 (s, 1H), 2.75 (s, 3H),

Step 4: To a solution of compound 4 (45 g, 254 mmol, 1.0 eq) in DMF (250mL) was added NaH (12.2 g, 305 mmol, 60% purity, 1.2 eq) at 0° C. inportion. The mixture was stirred at 0° C. for 1 h. Then the mixture waswarmed to 25° C. and SEM-Cl (50.8 g, 304.8 mmol, 54 mL, 1.2 eq) wasadded in dropwise within 1 h. The mixture was stirred at 25° C. for 3 h.LCMS showed the reaction was completed. All the mixture was poured intoice-water and combined extracted with ethyl acetate (1 L*2). Thecombined organic phase was washed with brine (500 mL*N), dried withanhydrous Na₂SO₄, filtered and concentrated in vacuum. The mixture waspurified by silica gel (Petroleum ether:Ethyl acetate=10:1 to 5:1,Rf=0.32) to give compound 5 (95 g, 309 mmol, 40.6% yield) as yellowsolid. ¹H NMR: (400 MHz, CDCl₃) δ 8.70 (s, 1H), 7.49 (d, J=3.2 Hz, 1H),6.68 (d, J=4.0 Hz, 1H), 5.74 (s, 2H), 3.61 (t, J=8.0 Hz, 2H), 2.99 (s,3H), 0.99 (t, J=8.4 Hz, 2H), 0.00 (s, 9H)

Step 5: To a solution of compound 5 (32 g, 104 mmol, 1.0 eq) and Pd/C(3.2 g) in THF (300 mL) was added AcOH (31.3 g, 520.5 mmol, 30 mL, 5.0eq), the suspension was degassed under vacuum and purged with H₂ threetimes. The mixture was stirred under H₂ (50 psi) at 30° C. for 12 hours.LCMS showed the reaction was completed. The mixture was filtered andconcentrated in vacuum. The residue was purified by silica gel(Petroleum ether:Ethyl acetate=10:1 to 2:1, Rf=0.2) to give compound 6(53 g, 191 mmol, 61.2% yield) as yellow solid. ¹H NMR: (400 MHz, CDCl₃)δ 7.25 (d, J=4.0 Hz, 1H), 6.37 (d, J=2.8 Hz, 1H), 5.67 (d, J=4.4 Hz,2H), 3.61 (t, J=8.4 Hz, 2H), 3.48 (s, 2H), 2.58 (s, 3H), 0.99 (t, J=8.4Hz, 2H), 0.00 (s, 9H)

Step 6: To a solution of compound 6 (14.3 g, 51.5 mmol, 1.0 eq) intoluene (250 mL) was added Ac₂O (20 g, 195.9 mmol, 18 mL, 3.8 eq) andAcOK (20.2 g, 206.2 mmol, 4.0 eq), the mixture was stirred at 60° C. for3 h. Then isopentyl nitrate (17.2 g, 129 mmol, 2.5 eq) was added to themixture in dropwise, the mixture was heated to 110° C. and stirred for 9h. TLC (Petroleum ether:Ethyl acetate=2:1, Rf=0.1) showed the reactionwas completed. The two mixtures was cooled to 25° C. and poured intowater (500 mL). The aqueous phase was extracted with ethyl acetate (200mL*2). The combined organic phase was washed with brine (200 mL), driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuum. The residuewas purified by silica gel (Petroleum ether:Ethyl acetate=10:1 to 2:1)to give compound 7 (20 g, 60.5 mmol, 58.7% yield) as yellow solid. ¹HNMR: (400 MHz, CDCl₃) δ 9.01 (s, 1H), 8.44 (s, 1H), 7.64 (d, J=3.6 Hz,1H), 6.77 (d, J=3.9 Hz, 1H), 5.82 (d, J=6.4 Hz, 2H), 3.65 (t, J=8.4 Hz,3H), 2.90 (s, 2H), 1.01 (t, J=8.4 Hz, 2H), 0.00 (s, 9H).

Step 7: To a solution of compound 7 (20 g, 60.5 mmol, 1.0 eq) in MeOH(250 mL) was added K₂CO₃ (16.7 g, 121.0 mmol, 2.0 eq), the mixture wasstirred at 20° C. for 5 h. LCMS showed the reaction was completed. Themixture was concentrated in vacuo and water (100 mL) was added. Theaqueous phase was extracted with ethyl acetate (100 mL*3). The combinedorganic phase was washed with brine (100 mL*), dried with anhydrousNa₂SO₄, filtered and concentrated in vacuum. The residue was purified bysilica gel (Petroleum ether:Ethyl acetate=10:1 to 2:1, Rf=0.1) to givecompound IM-14-7 (17 g, 58.9 mmol, 97.4% yield) as yellow solid. ¹H NMR(400 MHz, CDCl₃) δ 10.62 (s, 1H), 8.43 (s, 1H), 8.06 (s, 1H), 7.60 (d,J=3.6 Hz, 1H), 6.66 (d, J=3.6 Hz, 1H), 5.82 (s, 21H), 3.65-3.69 (m, 2H),1.09 (s, 2H), 0.00 (s, 9H).

EXAMPLE 3 Alternative Preparation of IM-14-3

General procedure for preparation of compound 2: To a solution of5-bromo-6-methyl-1H-pyrrolo[2,3-b]pyridine (10 g, 47.4 mmol, 1.00 eq) inDMF (150.00 mL), the mixture was cooled to 0° C. and NaH (2.84 g, 71mmol, 60% purity, 1.50 eq) was added to the mixture. The reactionmixture was stirred at 0° C. for 1 hr,2-(chloromethoxy)ethyl-trimethyl-silane (10.3 g, 61.6 mmol, 11 mL, 1.30eq) was added dropwise to the mixture. The mixture was stirred at 25° C.for 3hr. TLC indicated 0% of Reactant 1 was remained, and one major newspot with larger polarity (Petroleum ether:Ethyl acetate=5:1, Rf=0.51)was detected. The mixture was poured into NH₄Cl solution (40 ml),concentrated, then extracted with EA (30 ml×3), combine the organiclayers, washed with brine (20 ml), and concentrated to give crudeproduct. The residue was purified by column chromatography (SiO2,Petroleum ether/Ethyl acetate=100/1 to 5:1). Compound 2 (10.00 g, 29.30mmol, 61.84% yield) was obtained. ¹H NMR: ET6225-38-P1A 400 MHz CDCl₃δ8.01 (s, 1H), 7.26 (d, J=3.2 Hz, 1H), 6.40 (d, J=3.2 Hz, 1H), 5.62 (s,2H), 3.55 (t, J=16 Hz, 2H), 2.75 (s, 3H), 0.91 (t, J=16 Hz, 2H), 0.07(s, 9H).

General procedure for preparation of compound 3: A mixture of compound 2(5.50 g, 16.1 mmol, 1.00 eq), diphenylmethanimine (3.50 g, 19.3 mmol,1.20 eq), Xantphos (1.86 g, 3.22 mmol, 0.20 eq), Cs₂CO₃ (10.5 g, 32.2mmol, 2.00 eq) in dioxane (150 mL) was degassed and purged with N₂ for 3times, and then Pd₂(dba)₃ (885 mg, 966 umol, 0.06 eq) was added to themixture. Then the mixture was stirred at 100° C. for 15 hour under N₂atmosphere. TLC (Petroleum ether/Ethyl acetate=5:1) showed many spot.The desired compound was detected by LCMS (RT=1.363). The reactionmixture was concentrated under reduced pressure to give a residue. Thecrude product 3 was used into the next step without further purification

General procedure for preparation of compound 4: A mixture of crudecompound 3 (8.00 g, 11.8 mmol, 1.00 eq), 0.5 N HCl (100 mL), in THF (100mL) was degassed and purged with N₂ for 3 times, and then the mixturewas stirred at 25° C. for 14 hour under N₂ atmosphere. TLC (Petroleumether/Ethyl acetate=3:1) showed the desired product was detected. Themixture was quenched by aq. NaHCO₃ (200 ml) to adjust pH=8, thenextracted with Ethyl acetate (100 ml×3), combine the organic layers,washed with brine(100 ml), dried over sodium sulfate, concentrated togive crude product. The residue was purified by column chromatography(SiO2, Petroleum ether/Ethyl acetate=100/1 to 5:1) Compound 4 (3 g, 8.65mmol, 73.5% yield, 80% purity) was obtained as a solid. ¹H NMR:ET6225-46-P1A 400 MHz CDCl₃ δ 7.25 (s, 2H), 6.42 (s, 1H), 5.67 (s, 2H),3.60 (t, J=8 Hz, 2H), 2.62 (s, 3H), 0.98 (t, J=8 Hz, 2H), 0.04 (s, 9H).

General procedure for preparation of compound 5: A mixture of compound 4(2.00 g, 7.21 mmol, 1.00 eq) and Ac₂O (1.6 g, 15.6 mmol, 2.2 eq) inCHCl₃ (50.00 mL) was added AcOK (7.08 g, 72.10 mmol, 10 eq), and thenthe mixture was stirred at 25° C. for 3 hr, under N₂ atmosphere. thenisoamyl nitrate (3.38 g, 28.8 mmol, 4 eq) was added, heated to 60° C.for 14 hr, TLC(Petroleum ether/Ethyl acetate=3:1, Rf (product=0.43)showed the starting material was consumed completely. The mixture wasquenched by aq. NaHCO₃ (200 ml), then extracted with DCM (100ml×3),combine the organic layers, washed with brine (100 ml), dried oversodium sulfate, concentrated to give crude product. The residue waspurified by column chromatography (SiO2, Petroleum ether/Ethylacetate=100/1 to 5:1). Compound 5_Ac (600 mg, 21.36% yield, 85% purity)andN-[6-methyl-1-(2-trimethylsilylethoxymethyl)pyrrolo[2,3-b]pyridin-5-yl]acetamide(1.50 g, 4.70 mmol, 65.12% yield) were obtained.

A mixture of Compound 5_Ac (600 mg, 1.82 mmol, 1.00 eq) in MeOH (5 mL)was added K₂CO₃ (753 mg, 5.45 mmol, 3.00 eq), and then the mixture wasstirred at 25° C. for 2 hr, under N₂ atmosphere. TLC showed none ofCompound 5 was remained. The mixture was poured into water (200 ml), andthen extracted with DCM (100 ml×3), combine the organic layers, washedwith brine (100 ml), and dried over sodium sulfate, concentrated to givecrude product. The residue was purified by column chromatography (SiO2,Petroleum ether/Ethyl acetate=100/1 to 5:1). Compound 5 (500 mg, 95.25%yield) was obtained as a yellow solid. ¹H NMR: ET6225-47-P1A 400 MHzCDCl₃6 9.05 (s, 1H), 8.45 (s, 1H), 7.64 (s, 1H), 6.77(s, 1H), 5.82(s,2H), 3.65 (t, J=8 Hz, 2H), 2.90(s, 3H), 1.01 (t, J=8 Hz, 2H), 0.01 (s,9H). ¹H NMR: ET6225-51-P1A 400 MHz CDCl₃ δ 8.05 (s, 1H), 7.60 (s, 1H),6.66(s, 1H), 5.82(s, 2H), 3.67 (t, J=16 Hz, 2H), 1.01 (t, J=16 Hz, 2H),0.04 (s, 9H).

EXAMPLE 4 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

A mixture of compound IM-14-3 (500 mg, 1.73 mmol, 1.00 eq), ethyl4-bromo-2-fluoro-benzoate (641 mg, 2.60 mmol, 1.50 eq), Cs₂CO₃ (1.69 g,5.19 mmol, 3.00 eq) in DMF (10 mL) was degassed and purged with N₂ for 3times, and then the mixture was stirred at 125° C. for 1 hour under N₂atmosphere under microwave condition. TLC indicated the desired compound(Rf: 0.24). The reaction mixture was filtered and the filtrate wasconcentrated under reduced pressure to give a residue. The residue waspurified by silica gel column chromatography (Petroleum ether/Ethylacetate=100:1 to 3:1). ethyl4-bromo-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate(400 mg, 659 umol, 38.1% yield, 85% purity) was obtained as a yellowoil. ¹H NMR: ET6225-63-P1A 400 MHz CDCl₃ δ8.50 (s, 1 H) 7.90-7.96 (m, 3H) 7.72 (d, J=8.38 1 H) 7.60 (d, J=3.53 Hz, 1 H) 6.60 (d, J=4 Hz, 1 H)5.81 (s, 2 H) 3.97-4.07 (m, 2 H) 3.62-3.70 (m, 2 H) 0.97-1.05 (m, 2 H)0.83 (t, J=7.28 Hz, 3 H) 0.01 (s, 9 H).

A mixture of ethyl4-bromo-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate(800 mg, 1.55 mmol, 1.00 eq), compound 7 (594 mg, 1.86 mmol, 1.20 eq),Xantphos (359 mg, 621 umol, 0.40 eq), Cs₂CO₃ (1.52 g, 4.66 mmol, 3.00eq) in dioxane (20 mL) was degassed and purged with N₂ for 3 times, andthen Pd₂(dba)₃ (284 mg, 310 umol, 0.20 eq) was added to the mixture, themixture was stirred at 100° C. for 15 hour under N₂ atmosphere. TLCshowed the reaction was completed. The desired compound (Rf: 0.24) wasdetected. The reaction mixture was filtered and the filtrate wasconcentrated under reduced pressure to give a residue. The residue waspurified by silica gel chromatography (100-200 mesh silica gel,Petroleum ether/Ethyl acetate=100/1-3/1). Ethyl4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate(600 mg, 637 umol, 41.1% yield, 80% purity) was obtained as a yellowsolid. ¹H NMR: ET6225-84-p1a 400 MHz CDCl₃ δ 8.46 (s, 1 H) 8.04 (d,J=8.8 Hz, 1 H) 7.73-7.86 (m, 1 H) 7.55 (d, J=3.5 Hz, 1 H) 6.95-7.05 (m,4 H) 6.57 (d, J=3.5 Hz, 1 H) 5.80 (s, 2 H) 3.88 (q, J=7 Hz, 2 H)3.61-3.70 (m, 2 H) 3.37 (d, J=4.8 Hz, 4 H) 2.89 (s, 2 H) 2.42 (d, J=4.8Hz, 4 H) 2.29 (s, 2 H) 2.07 (s, 2 H) 1.63 (s, 7 H) 1.52 (t, J=6.39 Hz, 2H) 0.85-1.08 (m, 16 H) 0.68 (t, J=7.2 Hz, 3 H) 0.00 (s, 9 H).

A mixture of ethyl4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoate(500 mg, 664 umol, 1.00 eq), NaOH (79.6 mg, 1.99 mmol, 3.00 eq) in MeOH(10 mL), THF (10 mL), H₂O (5 mL) was stirred at 0-25° C. for 2 hourunder N₂ atmosphere. The desired compound was detected. The reactionmixture was concentrated under reduced pressure to give a residue. thisresidue was dissolved in water, then was adjusted to pH=4-5 with 0.5 MHCl and extracted with Ethyl acetate (20 ml×3) combine the organiclayers, washed with brine, dried over sodium sulfate, concentrated togive4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoicacid (400 mg, 74.8% yield, 90% purity) was obtained as a white solid. ¹HNMR: ET6225-85-p1a 400 MHz CDCl₃ δ8.45 (s, 1 H) 8.07 (d, J=9.2 Hz, 1 H)7.85 (s, 1 H) 7.53 (d, J=3.9 Hz, 1 H) 7.30-7.34 (m, 3 H) 7.00 (d, J=8.4Hz, 2 H) 6.84-6.93 (m, 2 H) 6.52-6.57 (m, 1 H) 5.76 (s, 2 H) 3.61-3.69(m, 2 H) 3.35 (s, 3 H) 2.47 (s, 2 H) 2.28 (s, 2 H) 2.06 (s, 2 H) 1.49(t, J=5.9 Hz, 2 H) 1.27-1.36 (m, 1 H) 0.96-1.05 (m, 9 H), 0.01 (s, 9 H).

A mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoicacid (400 mg, 551 umol, 1.00 eq), and HATU (629 mg, 1.65 mmol, 3.00 eq)NH₄Cl (147 mg, 2.76 mmol, 5.00 eq) Et₃N (558 mg, 5.51 mmol, 10.00 eq) inDMF (3 mL) was stirred at 25° C. for 15 hr under N₂ atmosphere. Thedesired compound was detected by TLC. The reaction mixture was filteredand the filtrate was concentrated under reduced pressure to give aresidue. The residue was purified by column chromatography (SiO2, DCM:MeOH=200/1 to 20:1).4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(300 mg, 75% yield) was obtained as a yellow solid. ¹H NMR:ET6225-86-p1a 400 MHz CDCl₃ δ8.44 (s, 1 H) 7.88-7.94 (m, 1 H) 7.83 (d,J=8.8 Hz, 1 H) 7.55-7.60 (m, 1 H) 7.39 (d, J=7.9 Hz, 2 H) 7.05 (d, J=7.9Hz, 2 H) 6.87 (s, 1 H) 6.65 (d, J=3.5 Hz, 1 H) 5.74-5.80 (s, 2 H) 3.66(m, 4 H) 3.53 (br. s., 2 H) 3.21-3.34 (m, 11 H) 2.09-2.32 (m, 3 H) 1.41(m, 17 H) 0.01 (s, 9 H).

A mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(300 mg, 414 umol, 1.00 eq) in THF (8 mL), then NaH (49.7 mg, 1.24 mmol,60% purity, 3.00 eq) was added at 0° C., stirred for 0.5 hr, then4-chloro-3-nitro-benzenesulfonyl chloride (117 mg, 456 umol, 1.10 eq) inTHF (2 mL) was added, the mixture was stirred at 25° C. for 2 hr underN₂ atmosphere. The desired compound was detected. The reaction mixturewas concentrated under reduced pressure to give a residue. The residuewas purified by prep-TLC (SiO2, DCM/MeOH=10/1).N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200.00 mg, 211.86 umol, 51.16% yield) was obtained as yellow solid. ¹HNMR: ET6215-89-p1a 400 MHz CDCl₃ δ8.44 (s, 1 H) 7.95 (s, 1 H) 7.76 (s, 1H) 7.40-7.53 (m, 1 H) 6.95 (d, J=7.8 Hz, 5 H) 6.75 (s, 1 H) 6.51 (s, 1H) 5.71 (s, 3 H) 5.30 (s, 2 H) 3.50-3.65 (m, 5 H) 3.26 (s, 3 H) 3.06 (d,J=6.65 Hz, 2 H) 2.85 (s, 1 H) 2.36 (s, 2 H) 2.21 (s, 1 H) 2.00 (s, 4 H)1.44 (s, 1 H) 1.14-1.31 (m, 8 H) 0.96 (m, 13 H) 0.07 (s, 9 H)

A mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200 mg, 212 umol, 1.00 eq) in TFA (3.00 mL) and DCM (3.00 mL) wasstirred at 25° C. for 14 hr under N₂ atmosphere. The desired compound(Rt=1.146) was detected on LC-MS. The reaction mixture was concentratedunder reduced pressure to give a crude as a yellow solid. A mixture ofthe crude (150 mg, 204 umol, 1.00 eq) and cyclohexylmethanamine (69.5mg, 614 umol, 3.00 eq) in CH₃CN (4 mL) in a microwave tube, thenDIPEA(79.3 mg, 614 umol, 3.00 eq) was added, the mixture was stirred at125° C. for 14 hr under N₂ atmosphere under microwave. The desiredcompound was detected by LCMS (Rt=0.573). The reaction mixture wasconcentrated under reduced pressure to give a residue. The residue waspurified by prep-HPLC (neutral) and the recovered product was purifiedby Re-prep-HPLC (TFA). Two batches of target compound were obtained asyellow solid. The first batch is 11 mg; the second batch is 23 mg. ¹HNMR: ET6225-91-p1c 400 MHz DMSO δ11.50 (br. s., 1 H) 8.64 (s, 1 H) 8.40(s, 1 H) 7.97 (s, 1 H) 7.89 (s, 1 H) 7.60-7.69 (m, 2 H) 7.56 (m, 1 H)7.40 (m, 2 H) 6.98-7.16 (m, 5 H) 6.35 (s, 1 H) 3.81-4.03 (m, 3 H)3.12-3.41 (m, 6 H) 2.24 (m, 2 H) 2.05 (s, 2 H) 1.65 (m, 3 H) 1.44-1.53(m, 2 H) 1.19-1.38 (m, 3 H) 0.96 (s, 6 H), LCMS: (M+H)⁺: 892.2.

EXAMPLE 6 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

To a mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5-methyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.00 g, 1.06 mmol, 1.00 eq) in dichloromethane (10 mL) was addedTrifluoroacetic Acid (15.40 g, 135 mmol, 10.00 mL) in one portion at 20°C. The mixture was stirred at 20° C. for 14 hrs, LCMS showed the productwas detected. The reaction mixture was concentrated under reducedpressure to remove dichloromethane, and then diluted with water (100mL), and extracted with ethyl acetate (20 mL*3). The combined organiclayers were washed with brine (100 mL), dried over Na₂SO₄, filtered andconcentrated under reduced pressure to give the crude product CompoundN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(800.00 mg, crude) was used into the next step without furtherpurification.

A mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(100 mg, 118 umol, 1.00 eq) and 1-tetrahydropyran-4-ylpiperidin-4-amine(65.5 mg, 355 umol, 3.00 eq) in CH₃CN (1.0 mL) in a microwave tube, thenDIPEA (153 mg, 1.19 mmol, 206 uL, 10 eq) was added, the mixture wasstirred at 125° C. for 14 hr under N₂ atmosphere under microwave. LCMSand HPLC showed the product was detected. The reaction mixture wasconcentrated under reduced pressure to remove CH₃CN, and then dilutedwith water (10 mL) and extracted with dichloromethane (5 mL*3). Thecombined organic layers were washed with brine (10 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (prep-HPLC (column:YMC-Actus Triart C18 150*30 5u; liquid phase: [A-10 mM NH₄HCO₃ in H₂O;B-ACN]B%: 45%-65%,12 min])) to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((1-(tetrahydro-2H-pyran-4-yl)piperidin-4-yl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(2.60 mg, 2% yield) as a white solid. LCMS: (M/2+1): 481.1 ¹H NMR: CDCl₃400 MHz δ 8.45 (s, 1 H) 8.22-8.38 (m, 2 H) 7.81 (d, J=8.0 Hz, 1 H) 7.54(s, 2 H) 7.43 (d, J=8.0 Hz, 1 H) 6.87 (d, J=8.0 Hz, 4 H) 6.67 (s, 1 H)6.31 (s, 1 H) 3.98 (d, J=8.0 Hz, 2 H) 3.29-3.35 (m, 3 H) 3.20 (s, 5 H)2.74 (s, 3 H) 2.25 (s, 5 H) 2.14 (s, 3 H) 1.92 (s, 5 H) 1.18 (s, 2 H)0.89 (s, 9 H)

EXAMPLE 7 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((3-morpholinopropyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

A mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (100 mg, 118 umol, 1.00 eq) and3-morpholinopropan-1-amine (51 mg, 355 umol, 3.00 eq) in CH₃CN (1.00 mL)in a microwave tube, then DIPEA (60 mg, 464 umol, 3.92 eq) was added,the mixture was stirred at 125° C. for 14 hr under N₂ atmosphere undermicrowave. LCMS and HPLC showed the product was detected. The reactionmixture was concentrated under reduced pressure to remove CH₃CN, andthen diluted with water (10 mL) and extracted with dichloromethane (5mL*3). The combined organic layers were washed with brine (10 mL), driedover Na₂SO₄, filtered and concentrated under reduced pressure to give aresidue. The residue was purified by prep-HPLC (prep-HPLC (column:YMC-Actus Triart C18 150*30 5u; liquid phase: [A-10mM NH₄HCO₃ in H₂O;B-ACN]B%: 45%-75%,12 min])) to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((3-morpholinopropyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(2.0 mg, 2% yield) as a yellow solid. LCMS: (M/2+1): 461.1 ¹H NMR:ET10360-22-P1A1 CDCl₃ 400 MHz δ 9.06 (s, 1 H) 8.46 (s, 1 H) 8.23-8.39(m, 2 H) 7.81 (d, J=8.0 Hz, 1 H) 7.50-7.59 (m, 2 H) 7.34 (s, 1 H)6.80-6.92 (m, 3 H) 6.68 (s, 1 H) 6.46 (d, J=8.0 Hz, 1 H) 6.28 (s, 1 H)3.64-3.78 (m, 4 H) 3.12-3.29 (m, 6 H) 2.66-2.84 (m, 4 H) 2.44 (s, 7 H)2.26 (s, 4 H) 2.08-2.17 (m, 3 H) 1.92 (s, 2 H) 1.30-1.43 (m, 4 H)1.06-1.21 (m, 4 H) 0.89 (s, 7 H)

EXAMPLE 8 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200.00 mg, 211.86 μmol, 1.00 eq) was dissolved in DCM (3.00 mL), towhich TFA (4.62 g, 40.52 mmol, 3.00 mL, 191.25 eq) was added in oneportion. The mixture was then stirred at 25° C. under N₂ atmosphere for14 h. After removal of the solvent,N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(180.00 mg, crude) was obtained as a dark yellow solid which wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

To the mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(170.00 mg, 201.47 μmol, 1.00 eq) and morpholine (52.66 mg, 604.41 μmol,53.19 μL, 3.00 eq) in CH₃CN (4.00 mL), DIEA (78.12 mg, 604.41 μmol,105.57 μL, 3.00 eq) was added. The resulting mixture was taken up into amicrowave tube, and heated under microwave at 80° C. under N₂ atmospherefor 2 h. After removal of the solvent, the crude4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (300.00 mg, crude) was obtained as a black brown oilwhich was purified together with EW5403-10 by Prep-HPLC (HCl asadditive) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-morpholino-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt (41.40 mg) as a yellow solid. The product was confirmed by ¹HNMR and LC-MS.

EXAMPLE 9 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of Tert-butyl 3-morpholinoazetidine-1-carboxylate A mixture oftert-butyl 3-oxoazetidine-1-carboxylate (3.50 g, 20.45 mmol, 1.00 eq)and morpholine (2.32 g, 26.59 mmol, 2.34 mL, 1.30 eq) in DCE (250.00 mL)was stirred at 30° C. for 1 h, and then NaBH(OAc)₃ (5.63 g, 26.59 mmol,1.30 eq) was added. The mixture was stirred at 30° C. for additional 15h. Sat. Na₂CO₃ solution (200 mL) was added to the mixture, and themixture was stirred at 30° C. for 10 min The organic layer wasseparated, washed with H₂O (200 mL) and brine (200 mL), and thenconcentrated under reduced pressure. The residue was purified by silicagel chromatography eluted with PE to PE:EA=1:1 to give tert-butyl3-morpholinoazetidine-1-carboxylate (4.50 g, 18.57 mmol, 90.81% yield)as a light yellow oil which was confirmed by ¹H NMR.

Synthesis of 4-(azetidin-3-yl)morpholine TFA salt: A mixture oftert-butyl 3-morpholinoazetidine-1-carboxylate (500.00 mg, 2.06 mmol,1.00 eq) and TFA (7.70 g, 67.53 mmol, 5.00 mL, 32.78 eq) in DCM (20.00mL) was stirred at 30° C. for 15 h. After removal of the solvent,4-(azetidin-3-yl)morpholine TFA salt (280.00 mg, crude) was obtained asa yellow oil which was confirmed by LC-MS used directly without furtherpurification.

Synthesis ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200.00 mg, 211.86 μmol, 1.00 eq) was dissolved in DCM (3.00 mL), towhich TFA (4.62 g, 40.52 mmol, 3.00 mL, 191.25 eq) was added in oneportion. The resulting mixture was then stirred at 25° C. under N₂atmosphere for 14 h. After removal of the solvent,N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200.00 mg, crude) was obtained as a dark yellow solid which wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt: A mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(78.37 mg, 551.10 μmol, 3.00 eq) in CH₃CN (4.00 mL) was added DIEA(71.22 mg, 551.10 μmol, 96.24 μL, 3.00 eq). The resulting mixture wastaken up into a microwave tube and heated using microwave at 80° C. for2 h under N₂ atmosphere. After removal of the solvent,4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-(3-morpholinoazetidin-1-yl)-3-nitro-phenyl]sulfonyl-2-(5H-pyrazolo[BLAH]pyrrolo[BLAH]-pyridin-1-yl)benzamide(300.00 mg, crude) was obtained as a black brown oil which was purifiedtogether with EW5403-7 by Prep-HPLC (HCl) and then by SFC (column:Phenomenex Gemini 150*25mm*10 um; mobile phase: [water (0.05% ammoniahydroxide v/v)-ACN]; B%: 35%-65%, 10 min) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(3-morpholinoazetidin-1-yl)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt (2.40 mg, 2.51 μmol, 7.69% yield, HCl) as a yellow solid. Theproduct was confirmed by LC-MS and ¹H NMR.

EXAMPLE 10 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(100.00 mg, 105.93 μmol, 1.00 eq) was dissolved in DCM (3.00 mL), towhich TFA (2.31 g, 20.26 mmol, 1.50 mL, 191.25 eq) was added in oneportion. The resulting mixture was then stirred at 25° C. under N₂atmosphere for 14 h. After removal of the solvent,N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(101.00 mg, crude) was obtained as a dark yellow solid which wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide.

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt: The mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(101.00 mg, 119.70 μmol, 1.00 eq) and 7-oxa-2-azaspiro[3.5]nonane (78.00mg, 359.10 μmol, 3.00 eq, OXALIC ACID salt) were dissolved in CH₃CN(4.00 mL), to which DIEA (92.82 mg, 718.20 μmol, 125.43 μL, 6.00 eq) wasadded. The resulting mixture was taken up into a microwave tube. Thetube was heated under N₂ atmosphere at 80° C. for 3 h. After removal ofthe solvent, the residue was purified by Prep-HPLC (HCl, column:Phenomenex Synergi C₁₈ 150*25*10 μm; mobile phase:[water(0.05%HCl)-ACN]; B%: 35%-55%, 7.8 min) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(7-oxa-2-azaspiro[3.5]nonan-2-yl)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(6.10 mg, 6.48 μmol, 5.42% yield, HCl) as a yellow solid. The productwas confirmed by LC-MS and ¹H NMR.

EXAMPLE 11 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of S-(tetrahydropyran-4-ylmethyl) ethanethioate:

A mixture of 4-(bromomethyl)tetrahydropyran (300.00 mg, 1.68 mmol, 1.00eq) and potassium thioacetate (575.62 mg, 5.04 mmol, 3.00 eq) weredissolved in DMF (10.00 mL). The resulting mixture was then stirred at25° C. for 3 h under N₂ atmosphere. The reacting solution was quenchedwith sat. aq. NaHCO₃ solution (50 mL) and extracted with DCM (50 mL×3).The combined organic layers were concentrated under reduced pressure toafford S-(tetrahydropyran-4-ylmethyl) ethanethioate (245.00 mg, 1.41mmol, 83.69% yield) as black brown oil. The product was confirmed byLC-MS and used directly for the next step without further purification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

The mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(100.00 mg, 105.93 82 mol, 1.00 eq) and S-(tetrahydropyran-4-ylmethyl)ethanethioate (55.38 mg, 317.79 umol, 3.00 eq) were dissolved in CH₃CN(4.00 mL), to which t-BuOK (59.43 mg, 529.65 μmol, 5.00 eq) was added inone portion. The resulting mixture was taken up into a microwave tubeand heated under N₂ atmosphere at 80° C. for 2 h. The reacting solutionwas poured onto silica gel chromatography and eluted with pure PE toEA:MeOH=5:1. The eluent was concentrated under reduced pressure, and theresidue was suspended in MeOH (4 mL) and stirred for 30 min Theprecipitate was collected to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(70.00 mg, crude) as an off-white solid. The crude product was confirmedby LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(30.00 mg, 28.85 μmol, 1.00 eq) was dissolved in DCM (5.00 mL), to whichTFA (13.86 g, 121.53 mmol, 9.00 mL, 4211.98 eq) was added. The resultingmixture was then stirred at 25° C. for 14 h. After removal of thesolvent, the residue was then dissolved in CH₃CN (5.00 mL), to whichDIEA (111.87 mg, 865.58 μmol, 151.17 μL, 30.00 eq) was added in oneportion. The resulting mixture was then stirred at 80° C. for 3 h, andthen the solvent and excess reagent were removed under reduced pressureto afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(60.00 mg, crude) as a black brown oil which was purified together withEW5403-56 by Prep-HPLC (HCl) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((tetrahydro-2H-pyran-4-yl)methyl)thio)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt (21.40 mg, 22.62 μmol, 29.39% yield, HCl) as a yellow solid.The product was confirmed by LC-MS and ¹H NMR.

EXAMPLE 12 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile:

A mixture of1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile (8.44g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40 eq),Pd(dba)₂ (3.24 g, 5.63 mmol, 0.20 eq) and Cs₂CO₃ (27.51 g, 84.42 mmol,3.00 eq) in dioxane (300.00 mL) was stirred at 100° C. for 16 h under N₂atmosphere. After removal of the solvent, the residue was purified bysilica gel chromatography eluted with PE to PE:EA=5:1 to give4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 66.53% yield) as a yellow oil which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile:

A mixture of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 1.00 eq),5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine(5.40 g, 18.72 mmol, 1.00 eq) and Cs₂CO₃ (12.20 g, 37.44 mmol, 2.00 eq)in DMF (100.00 mL) was stirred at 120° C. for 3 h. After cooling to 15°C., the mixture was filtered. The filtrate was concentrated underreduced pressure. The residue was purified by silica gel chromatographyeluted with PE to PE:EA=3:1 to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(9.60 g, 13.59 mmol, 72.60% yield) as a black oil which was confirmed byLC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(2.60 g, 3.68 mmol, 1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH(30.00 mL), KOH (412.97 mg, 7.36 mmol, 2.00 eq) in H₂O (10.00 mL) wasadded. After addition, H₂O₂ (3.34 g, 29.44 mmol, 2.83 mL, 30% purity,8.00 eq) was added dropwise. The mixture was stirred at 25° C. for 14 h.H₂O (100 mL) was added to the mixture, and the mixture was extractedwith EA (100 mL×3). The combined organic layers were washed with H₂O(500 mL), and concentrated under reduced pressure. The residue waspurified by reverse phase column with TFA as additive to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.20 g, 1.66 mmol, 45.01% yield) as a yellow solid which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(500.00 mg, 690.22 umol, 1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52mL, 2.20 eq) in THF was added at 0° C. The mixture was stirred at 0° C.for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl chloride (181.92mg, 759.24 umol, 1.10 eq) was added. The mixture was stirred at 25° C.for 14 h. The reacting solution was quenched with sat. aq. NH₄Cl (50 mL)and extracted with EA (50 mL×3). The combined organic layers wereconcentrated under reduced pressure. The residue was purified by silicagel chromatogaphy (pure PE to DCM:MeOH=10:1) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(90.00 mg, 97.03 μmol, 14.06% yield) as a yellow oil. The product wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

A mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(20.00 mg, 21.56 μmol, 1.00 eq),(4-fluorotetrahydropyran-4-yl)-methanamine (3.99 mg, 29.97 μmol, 1.39eq) and DIEA (27.86 mg, 215.60 μmol, 37.65 μL, 10.00 eq) in DMF (5.00mL) was stirred at 50° C. for 14 h. The reacting solution was dilutedwith water (20 mL) and extracted with DCM (50 mL×3). The combinedorganic layers were concentrated under reduced pressure. The residue waspurified by silica gel chromatography(pure PE to PE:EA=1:2) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(40.00 mg, crude) as a yellow oil. The crude product was confirmed byLC-MS and used directly for the next step without further purification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(40.00 mg, 38.44 μmol, 1.00 eq) was dissolved in DCM (5.00 mL), to whichTFA (43.83 mg, 384.40 μmol, 28.46 μL, 10.00 eq) was added in oneportion. The resulting mixture was then stirred at 25° C. for 14 h.After removal of the solvent, crude4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(30.00 mg, crude, TFA) was obtained as a yellow oil which wasre-dissolved in MeCN (10.00 mL). To the solution, DIEA (36.77 mg, 284.50μmol, 49.69 μL, 10.00 eq) was added in one portion. The resultingmixture was then stirred at 80° C. for 2 h. The solvent and excessreagent were removed under reduced pressure. The residue was purified byPrep-HPLC (HCl) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-4(4-fluorotetrahydro-2H-pyran-4-yl)methyl)amino)-3-nitrophenyesulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt (6.90 mg, 7.29 μmol, 25.61% yield, HCl) as a light yellow solidwhich was confirmed by LC-MS and ¹H NMR.

EXAMPLE 13 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

The mixture ofN-((4-chloro-3-nitrophenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(200.00 mg, 211.86 μmol, 1.00 eq) and (1-methyl-4-piperidyl)methanamine(81.49 mg, 635.58 μmol, 3.00 eq) were dissolved in CH₃CN (10.00 mL), towhich DIEA (82.14 mg, 635.58 μmol, 111.00 μL, 3.00 eq) was added in oneportion. The resulting mixture was taken up into a microwave tube andheated at 80° C. under N₂ atmosphere for 2 h. The reacting solution waspoured onto silica gel chromatography and eluted with pure DCM toDCM:MeOH=5:1 to afford crude4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(50.00 mg, 48.27 μmol, 22.79% yield) as a yellow oil which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide:

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(50.00 mg, 48.27 μmol, 1.00 eq) was dissolved in DCM (10.00 mL), towhich TFA (16.51 mg, 144.81 μmol, 10.72 μL, 3.00 eq) was added. Theresulting mixture was then stirred at 25° C. for 1 h. The solvent andexcess reagent was removed under reduced pressure to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide(40.00 mg, crude) as a yellow oil which was used directly in the nextstep.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt: The mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)benzamide(40.00 mg, 42.76 μmol, 1.00 eq) and DIEA (16.58 mg, 128.28 μmol, 22.41μL, 3.00 eq) in CH₃CN (10.00 mL) was stirred at 80° C. for 5 h. Afterremoval of the solvent, the residue was purified by Prep-HPLC (HCl asadditive) and SFC to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamideHCl salt (23.60 mg, 26.06 μmol, 92.19% yield) as a light yellow solidwhich was confirmed by LC-MS and ¹H NMR.

EXAMPLE 14 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of 9H-fluoren-9-ylmethyl4-[tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate:The mixture of tert-butyl N-[4-fluoro-4-piperidyl)methyl]carbamate(150.00 mg, 645.74 μmol, 1.00 eq) and 9H-fluoren-9-ylmethylcarbonochloridate (250.58 mg, 968.61 μmol, 1.50 eq) were dissolved inTHF (20.00 mL) and H₂O (4.00 mL), to which NaHCO₃ (162.75 mg, 1.94 mmol,75.35 μL, 3.00 eq) was added in one portion. The resulting mixture wasthen stirred at 15° C. for 14 h. The reacting solution was diluted withwater (50 mL) and extracted with EA (50 mL×3). The combined organiclayers were concentrated under reduced pressure. The residue waspurified by silica gel chromatography (pure PE to PE:EA =5:1) to afford9H-fluoren-9-ylmethyl4-[(tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate(163.00 mg, 358.61 μmol, 55.54% yield) as an off-white solid. Theproduct was confirmed by LC-MS and used directly for the next stepwithout further purification.

Synthesis of 4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate TFA salt:9H-fluoren-9-ylmethyl4-[(tert-butoxycarbonylamino)methyl]-4-fluoro-piperidine-1-carboxylate(163.00 mg, 358.61 μmol, 1.00 eq) was dissolved in DCM (10.00 mL), towhich TFA (204.44 mg, 1.79 mmol, 132.76 μL, 5.00 eq) was added in oneportion. The resulting mixture was then stirred at 15° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure to afford9H-fluoren-9-ylmethyl 4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate(200.00 mg, crude, TFA) as a yellow oil. The crude product was useddirectly for the next step without further purification.

Synthesis of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile:A mixture of1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile (8.44g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40 eq),Pd(dba)₂ (3.24 g, 5.63 mmol, 0.20 eq) and Cs₂CO₃ (27.51 g, 84.42 mmol,3.00 eq) in dioxane (300.00 mL) was stirred at 100° C. for 16 h under N₂atmosphere. After removal of the solvent, the residue was purified bysilica gel chromatography eluted with PE to PE:EA=5:1 to give4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 66.53% yield) as a yellow oil which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile:A mixture of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 1.00 eq),5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine(5.40 g, 18.72 mmol, 1.00 eq) and Cs₂CO₃ (12.20 g, 37.44 mmol, 2.00 eq)in DMF (100.00 mL) was stirred at 120° C. for 3 h. After cooling to 15°C., the mixture was filtered. The filtrate was concentrated underreduced pressure. The residue was purified by silica gel chromatographyeluted with PE to PE:EA=3:1 to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(9.60 g, 13.59 mmol, 72.60% yield) as a black oil which was confirmed byLC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(2.60 g, 3.68 mmol, 1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH(30.00 mL), KOH (412.97 mg, 7.36 mmol, 2.00 eq) in H₂O (10.00 mL) wasadded. After addition, H₂O₂ (3.34 g, 29.44 mmol, 2.83 mL, 30% purity,8.00 eq) was added dropwise. The mixture was stirred at 25° C. for 14 h.H₂O (100 mL) was added to the mixture, and the mixture was extractedwith EA (100 mL×3). The combined organic layers were washed with H₂O(500 mL), and concentrated under reduced pressure. The residue waspurified by reverse phase column with TFA as additive to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.20 g, 1.66 mmol, 45.01% yield) as a yellow solid which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(500.00 mg, 690.22 μmol, 1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52mL, 2.20 eq) in THF was added at 0° C. The mixture was stirred at 0° C.for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl chloride (181.92mg, 759.24 μmol, 1.10 eq) was added. The mixture was stirred at 25° C.for 14 h. The reacting solution was quenched with sat. aq. NH₄Cl (50 mL)and extracted with EA (50 mL×3). The combined organic layers wereconcentrated under reduced pressure. The residue was purified by silicagel chromatogaphy (pure PE to DCM:MeOH=10:1) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(90.00 mg, 97.03 μmol, 14.06% yield) as a yellow oil. The product wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:Themixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide (250.00 mg, 269.53 μmol, 1.00 eq) and 9H-fluoren-9-ylmethyl4-(aminomethyl)-4-fluoro-piperidine-1-carboxylate (143.29 mg, 404.29μmol, 1.50 eq) were dissolved in DMF (50.00 mL), to which DIEA (348.34mg, 2.70 mmol, 470.73 μL, 10.00 eq) was added in one portion. Theresulting mixture was then stirred at 50° C. for 14 h. The reactingsolution was diluted with water (50 mL) and extracted with DCM (50mL×3). The combined organic layers were concentrated under reducedpressure to afford crude (9H-fluoren-9-yl)methyl4-(((4-(N-(4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenyeaminolmethyl)-4-fluoropiperidine-1-carboxylate(400.00 mg, crude) as a yellow oil which was re-dissolved in DMF (20.00mL). To the above solution, piperidine (134.95 mg, 1.58 mmol, 156.92 μL,5.00 eq) was added in one portion. The resulting mixture was thenstirred at 15° C. for 14 h. The reacting solution was poured onto silicagel chromatography and eluted with pure PE to DCM:MeOH=5:1 to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(268.00 mg, 257.76 μmol, 81.32% yield) as a yellow oil which wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(268.00 mg, 257.76 μmol, 1.00 eq) was dissolved in DCM (20.00 mL), towhich TFA (146.95 mg, 1.29 mmol, 95.42 μL, 5.00 eq) was added in oneportion. The resulting mixture was then stirred at 15° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(240.00 mg, crude) as a yellow oil which was confirmed by LC-MS and useddirectly for the next step without further purification.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoropiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(120.00 mg, 127.73 μmol, 1.00 eq) and oxetan-3-one (27.61 mg, 383.19μmol, 3.00 eq) were dissolved in MeOH (10.00 mL), to which NaBH₃CN(40.13 mg, 638.65 μmol, 5.00 eq) was added in one portion. The resultingmixture was then stirred at 15° C. for 14 h. The reacting solution wasdiluted with water (50 mL) and extracted with DCM (50 mL×3). Thecombined organic layers were concentrated under reduced pressure toafford crude4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-4(4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(120.00 mg, crude) as a yellow oil which was re-dissolved in CH₃CN(20.00 mL). To the above solution DIEA (77.89 mg, 602.70 μmol, 105.26μL, 5.00 eq) was added in one portion. The resulting mixture was thenstirred under N₂ atmosphere at 80° C. for 14 h. The reacting solutionwas diluted with water (50 mL) and extracted with DCM (50 mL×3). Thecombined organic layers were concentrated under reduced pressure. Theresidue was purified by Prep-TLC (EA/MeOH=10:1 and then EA:MeOH=5:1) toafford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(2.80 mg, 2.90 μmol, 2.41% yield) as a yellow solid. The product wasconfirmed by LC-MS and ¹H NMR.

EXAMPLE 15 Preparation of(R)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4(((4-(oxetan-3-yl)morpholin-2-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of 9H-fluoren-9-ylmethyl(2R)-2-[tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate: Amixture of tert-butyl N-[[(2S)-morpholin-2-yl]methyl]carbamate (64.00mg, 295.91 μmol, 1.00 eq), Fmoc-Cl (114.83 mg, 443.87 μmol, 1.50 eq) andNa₂CO₃ (94.09 mg, 887.74 μmol, 3.00 eq) in a mixture of THF (10.00 mL)and H₂O (2.00 mL) was stirred at 20° C. for 12 h. The mixture wasextracted with EA (20 mL×3), and the combined organic layers wereconcentrated under reduced pressure. The residue was purified byPrep-TLC (PE:EA=3:1) to give 9H-fluoren-9-ylmethyl(2R)-2-[(tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate(111.00 mg, 253.13 μmol, 85.54% yield) as a colorless oil which wasconfirmed by LC-MS.

Synthesis of 9H-fluoren-9-ylmethyl(2R)-2-(aminomethyl)morpholine-4-carboxylate TFA salt: A mixture of9H-fluoren-9-ylmethyl(2R)-2-[(tert-butoxycarbonylamino)methyl]morpholine-4-carboxylate(111.00 mg, 253.12 μmol, 1.00 eq) and TFA (577.21 mg, 5.06 mmol, 374.81μL, 20.00 eq) in DCM (10.00 mL) was stirred at 15° C. for 3 h. Afterremoval of the solvent, crude 9H-fluoren-9-ylmethyl(2R)-2-(aminomethyl)morpholine-4-carboxylate TFA salt (80.00 mg) wasobtained as a light yellow oil which was used directly in the next step.

Synthesis of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile:A mixture of1-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazine(2.54 g, 7.15 mmol, 1.00 eq, HCl), 4-bromo-2-fluoro-benzonitrile (8.44g, 42.21 mmol, 1.50 eq), Xantphos (6.51 g, 11.26 mmol, 0.40 eq),Pd(dba)₂ (3.24 g, 5.63 mmol, 0.20 eq) and Cs₂CO₃ (27.51 g, 84.42 mmol,3.00 eq) in dioxane (300.00 mL) was stirred at 100° C. for 16 h under N₂atmosphere. After removal of the solvent, the residue was purified bysilica gel chromatography eluted with PE to PE:EA=5:1 to give4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 66.53% yield) as a yellow oil which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile:A mixture of4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-fluoro-benzonitrile(8.20 g, 18.72 mmol, 1.00 eq),5-((2-(trimethylsilyl)ethoxy)methyl)-1,5-dihydropyrazolo[4,3-b]pyrrolo[3,2-e]pyridine(5.40 g, 18.72 mmol, 1.00 eq) and Cs₂CO₃ (12.20 g, 37.44 mmol, 2.00 eq)in DMF (100.00 mL) was stirred at 120° C. for 3 h. After cooling to 15°C., the mixture was filtered. The filtrate was concentrated underreduced pressure. The residue was purified by silica gel chromatographyeluted with PE to PE:EA=3:1 to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(9.60 g, 13.59 mmol, 72.60% yield) as a black oil which was confirmed byLC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzonitrile(2.60 g, 3.68 mmol, 1.00 eq) in a mixture of DMSO (30.00 mL) and EtOH(30.00 mL), KOH (412.97 mg, 7.36 mmol, 2.00 eq) in H₂O (10.00 mL) wasadded. After addition, H₂O₂ (3.34 g, 29.44 mmol, 2.83 mL, 30% purity,8.00 eq) was added dropwise. The mixture was stirred at 25° C. for 14 h.H₂O (100 mL) was added to the mixture, and the mixture was extractedwith EA (100 mL×3). The combined organic layers were washed with H₂O(500 mL), and concentrated under reduced pressure. The residue waspurified by reverse phase column with TFA as additive to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.20 g, 1.66 mmol, 45.01% yield) as a yellow solid which was confirmedby LC-MS.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:

To a solution of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(500.00 mg, 690.22 μmol, 1.00 eq) in THF (10.00 mL), LiHMDS (1 M, 1.52mL, 2.20 eq) in THF was added at 0° C. The mixture was stirred at 0° C.for 30 min, and then 4-fluoro-3-nitro-benzenesulfonyl chloride (181.92mg, 759.24 μmol, 1.10 eq) was added. The mixture was stirred at 25° C.for 14 h. The reacting solution was quenched with sat. aq. NH₄Cl (50 mL)and extracted with EA (50 mL×3). The combined organic layers wereconcentrated under reduced pressure. The residue was purified by silicagel chromatogaphy (pure PE to DCM:MeOH=10:1) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(90.00 mg, 97.03 μmol, 14.06% yield) as a yellow oil. The product wasconfirmed by LC-MS and used directly for the next step without furtherpurification.

Synthesis of(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyesulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:A mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(80.00 mg, 86.25 μmol, 1.00 eq), 9H-fluoren-9-ylmethyl(2R)-2-(aminomethyl)morpholine-4-carboxylate (67.13 mg, 198.38 μmol,2.30 eq) and DIEA (55.73 mg, 431.25 μmol, 75.31 μL, 5.00 eq) in DMF(10.00 mL) was stirred at 50° C. under N₂ atmosphere for 14 h. Thereacting solution was diluted with water (20 mL) and extracted with DCM(50 mL×3). The combined organic layers were concentrated under reducedpressure to afford (R)-(9H-fluoren-9-yl)methyl2-(((4-(N-(4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenyl)amino)methyl)morpholine-4-carboxylate(100.00 mg, crude) as a yellow oil. Next, (R)-(9H-fluoren-9-yl)methyl2-(((4-(N-(4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoyl)sulfamoyl)-2-nitrophenyl)amino)methyl)morpholine-4-carboxylate(130.00 mg, 104.34 μmol, 1.00 eq) was dissolved in DMF (20.00 mL), towhich piperidine (88.85 mg, 1.04 mmol, 103.31 μL, 10.00 eq) was added inone portion. The resulting mixture was then stirred at 15° C. for 14 h.The reacting solution was poured onto silica gel chromatography (pure PEto DCM:MeOH=5:1) to afford(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-l[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(100.00 mg, crude) as a yellow oil. The crude product was confirmed byLC-MS and used directly for the next step without further purification.

Synthesis of(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)benzamide:(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(100.00 mg, 97.68 μmol, 1.00 eq) was dissolved in DCM (20.00 mL), towhich TFA (111.38 mg, 976.84 μmol, 72.32 μL, 10.00 eq) was added in oneportion. The resulting mixture was then stirred at 15° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure to afford(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)benzamide(100.00 mg, crude, TFA) as a yellow oil. The crude product was useddirectly for the next step without further purification.

Synthesis of(R)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:(R)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of(S)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)benzamide(100.00 mg, 108.29 μmol, 1.00 eq) and oxetan-3-one (23.41 mg, 324.87μmol, 3.00 eq) were dissolved in MeOH (20.00 mL), to which NaBH₃CN(20.41 mg, 324.87 μmol, 3.00 eq) was added in one portion. The resultingmixture was then stirred at 15° C. under N₂ atmosphere for 14 h. Thereacting solution was diluted with water (20 mL) and extracted with DCM(50 mL×3). The combined organic layers were concentrated under reducedpressure to afford crude(R)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)methyl)amino)phenyl)sulfonyl)benzamide(100.00 mg, crude) as a yellow oil which was then suspended in CH₃CN(20.00 mL). To the above solution, DIEA (131.94 mg, 1.02 mmol, 178.30μL, 10.00 eq) was added in one portion. The resulting mixture was thenstirred at 80° C. for 14 h. The solvent and excess reagent were removedunder reduced pressure. The residue was purified by Prep-HPLC (neutral)to afford(R)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-(((4-(oxetan-3-yl)morpholin-2-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.10 mg, 1.16 μmol, 1.13% yield) as a yellow solid. The product wasconfirmed by LC-MS and ¹H NMR. 11 mg of crude product was also obtained.

EXAMPLE 16 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Synthesis of Tert-butyl((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate: A mixture oftert-butyl ((4-fluoropiperidin-4-yl)methyl)carbamate (330.00 mg, 1.42mmol, 1.00 eq) and aq. HCHO (37%, 172.88 mg, 2.13 mmol, 158.61 μL, 1.50eq) in CH₃CN (10.00 mL) was stirred at 30° C. for 30 min, and thenNaBH₃CN (178.47 mg, 2.84 mmol, 2.00 eq) and AcOH (104.88 mg, 1.75 mmol,99.89 μL, 1.23 eq) were added in portions. The mixture was stirred at15° C. for 13.5 h. The reacting solution was diluted with water (100 mL)and extracted with EA (100 mL×4). The combined organic layers wereconcentrated under reduced pressure and purified by silica gelchromatography (PE:EA =1:1 to EA:MeOH=5:1) to afford tert-butyl((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate (290.00 mg, 1.18mmol, 82.91% yield) as a colorless oil, which was confirmed by LC-MS.

Synthesis of (4-fluoro-1-methylpiperidin-4-yl)methanamine: Tert-butyl((4-fluoro-1-methylpiperidin-4-yl)methyl)carbamate (290.00 mg, 1.18mmol, 1.00 eq) was dissolved in DCM (10.00 mL), to which TFA (3.08 g,27.01 mmol, 2.00 mL, 22.89 eq) was added in portions. The resultingmixture was then stirred at 15° C. for 15 h. The solvent and excessreagent were removed under reduced pressure to afford(4-fluoro-1-methylpiperidin-4-yl)methanamine (320.00 mg, crude) as ayellow oil.

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-fluoro-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(140.00 mg, 150.94 μmol, 1.00 eq) and(4-fluoro-1-methylpiperidin-4-yl)methanamine (66.21 mg, 452.82 μmol,3.00 eq) were dissolved in DMF (20.00 mL), to which DIEA (195.07 mg,1.51 mmol, 263.61 μL, 10.00 eq) was added in one portion. The resultingmixture was then stirred under N₂ atmosphere at 50° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure. Theresidue was purified by silica gel chromatography (pure DCM toDCM:MeOH=3:1) to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(141.00 mg, 133.81 μmol, 88.65% yield) as a yellow oil, which wasconfirmed by LC-MS. LC-MS: EW5403-312-P1A00 (M+H⁺=1054, M/2+H⁺=527).

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-((2-(trimethylsilyl)ethoxy)methyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(141.00 mg, 133.81 μmol, 1.00 eq) was dissolved in DCM (10.00 mL), towhich TFA (152.57 mg, 1.34 mmol, 99.07 μL, 10.00 eq) was added in oneportion. The resulting mixture was then stirred at 15° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(150.00 mg, crude) as a yellow oil, which was confirmed by LC-MS. LC-MS:EW5403-317-P1Z (M+H⁺=953).

Synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(5-(hydroxymethyl)pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(150.00 mg, 157.31 μmol, 1.00 eq) and DIEA (203.31 mg, 1.57 mmol, 274.74μL, 10.00 eq) were dissolved in CH₃CN (20.00 mL). The resulting mixturewas then stirred at 80° C. under N₂ atmosphere for 14 h. The solvent andexcess reagent were removed under reduced pressure. The residue waspurified by column: Phenomenex Synergi C,₈ 150*25*10 μm; mobile phase:[water (0.05% HCl)-ACN]; B%: 42%-62%, 7.8 min to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-fluoro-1-methylpiperidin-4-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(1.8 mg, 1.95 μmol, 1.24% yield) as a yellow solid, which was confirmedby LC-MS and ¹H NMR. LC-MS: EW5403-318-P1B (M+H⁺=924, M/2+H⁺=462). ¹HNMR: EW5403-318-P1A0 400 MHz DMSO-d₆, δ 11.43 (brs, 1 H), 10.65 (brs, 1H), 8.54 (s, 1 H), 8.30 (s, 1 H), 8.05 (s, 1 H), 7.77 (s, 1 H),7.36-7.59 (m, 4 H), 7.07-7.09 (m, 3 H), 6.95-6.97 (m, 2 H), 6.30 (s, 1H), 3.71-3.77 (m, 2 H), 3.24-3.27 (m, 3 H), 3.03-3.17 (m, 3 H),2.87-2.89 (m, 2 H), 2.10-2.12 (m, 4 H), 2.21-2.50 (m, 4 H), 1.99 (s, 3H), 1.41-1.45 (m, 2 H), 1.16-1.23 (m, 6 H), 0.95 (s, 6 H).

EXAMPLE 17 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Step 1: Tert-butyl4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate:The mixture of 4-fluoro-3-nitro-benzenesulfonamide (300.00 mg, 1.36mmol, 1.00 eq) and tert-butyl4-fluoro-4-(hydroxymethyl)piperidine-1-carboxylate (317.26 mg, 1.36mmol, 1.00 eq) were dissolved in THF (50.00 mL), to which NaH (163.20mg, 4.08 mmol, 60% purity, 3.00 eq) was added at 0° C. in one portion.The resulting mixture was then stirred at 15° C. for 14 h. The reactingsolution was quenched with EtOH (10 mL), diluted with water (50 mL) andextracted with DCM (100 mL×3). The combined organic layers wereconcentrated under reduced pressure and purified together withEW5403-280-P1 by silica gel chromatography (pure PE to PE:EA =1:1) toafford tert-butyl4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate(560 mg, 1.29 mmol, 85.69% yield) as an off-white solid, which wasconfirmed by LC-MS. LC-MS: EW5403-286-P1A0 (M+Na⁺=456).

Step 2: 4-((4-fluoropiperidin-4-yl)methoxy)-3-nitrobenzenesulfonamideTert-butyl4-fluoro-4-((2-nitro-4-sulfamoylphenoxy)methyl)piperidine-1-carboxylate(100.00 mg, 230.71 μmol, 1.00 eq) was dissolved in DCM (20.00 mL), towhich TFA (131.53 mg, 1.15 mmol, 85.41 μL, 5.00 eq) was added in oneportion. The resulting mixture was then stirred at 15° C. for 14 h. Thesolvent and excess reagent were removed under reduced pressure to afford4-((4-fluoropiperidin-4-yl)methoxy)-3-nitrobenzenesulfonamide (80 mg,crude) as an off-white solid. LC-MS: EW5403-287-P1Z0 (M+H⁺=334).

Step 3:4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrobenzenesulfonamide:The mixture of4-[(4-fluoro-4-piperidyl)methoxy]-3-nitro-benzenesulfonamide (70.00 mg,156.47 μmol, 1.00 eq, TFA) and oxetan-3-one (67.65 mg, 938.82 μmol, 6.00eq) were dissolved in MeOH (10.00 mL), to which NaBH₃CN (98.33 mg, 1.56mmol, 10.00 eq) was added in one portion. The resulting mixture was thenstirred at 15° C. for 14 h. The solvent and excess reagent were removedunder reduced pressure. The residue was purified together withEW5403-288-P1 by silica gel chromatography (pure DCM to DCM:MeOH=10:1)to afford4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrobenzenesulfonamide(100 mg, crude) as a light yellow oil, which was confirmed by LC-MS.LC-MS: EW5403-289-P1A2 (M+H⁺=390).

Step 4: The synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:The mixture of4-[[4-fluoro-1-(oxetan-3-yl)-4-piperidyl]methoxy]-3-nitro-benzenesulfonamide(50.00 mg, 128.40 μmol, 1.00 eq),4-[4-[[2-(4-chlorophenyl)-4,4-dimethyl-cyclohexen-1-yl]methyl]piperazin-1-yl]-2-(5H-pyrazolo[BLAH]pyrrolo[BLAH]pyridin-1-yl)benzoicacid (76.42 mg, 128.40 μmol, 1.00 eq), DMAP (31.37 mg, 256.81 μmol, 2.00eq) and EDCI (49.23 mg, 256.81 μmol, 2.00 eq) were dissolved in DCM(20.00 mL). The resulting mixture was then stirred at 15° C. for 14 h.The reacting solution was poured onto silica gel chromatography andeluted with DCM to DCM:MeOH=5:1, and then purified by column: PhenomenexSynergi C18 150*25*10 μm; mobile phase: [water (0.05% HCl)-ACN]; B%:42%-62%, 7.8 min to afford4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-(oxetan-3-yl)piperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(8.4 mg, 8.69 μmol, 6.8% yield) as a light yellow solid, which wasconfirmed by LC-MS and ¹H NMR. LC-MS: EW5403-300-P1C0 (M/2+H⁺=483). ¹HNMR: EW5403-300-P1A0 400 MHz CD₃OD, δ 8.26 (s, 1 H), 8.18 (s, 2 H),7.99-8.00 (m, 1 H), 7.67-7.70 (m, 1 H), 7.65 (s, 1 H), 7.15-7.37 (m, 7H), 6.52 (s, 1 H), 4.44-4.49 (m, 2 H), 4.12-4.18 (m, 3 H), 3.95 (s, 2H), 3.54-3.82 (m, 8 H), 3.35 (m, 2 H), 2.90 (s, 3 H), 2.38-2.49 (m, 6H), 2.14 (s, 2 H), 1.60 (s, 2 H), 1.29 (s, 1 H), 1.03 (s, 6 H).

EXAMPLE 18 Preparation of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

Step 1: the synthesis of3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide: To asolution of (tetrahydro-2H-pyran-4-yl)methanol (263.68 mg, 2.27 mmol,1.00 eq) in THF (20.00 mL), NaH (272.40 mg, 6.81 mmol, 60% purity, 3.00eq) was added at 0° C. The mixture was stirred at 0° C. for 30 min, andthen 4-fluoro-3-nitrobenzenesulfonamide (500.00 mg, 2.27 mmol, 1.00 eq)was added. The mixture was stirred at 25° C. for 15 h, and then thereaction mixture was quenched by MeOH (10 mL). After removal of thesolvent, the residue was purified by silica gel chromatography elutedwith PE:EA=5:1 to 1:1 to give3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide (446.00mg, 1.41 mmol, 62.11% yield) as yellow solid which was confirmed byLC-MS. LC-MS: EW6259-87-P1A2 (M+H⁺=317.0).

Step 2, the synthesis of4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide:To a mixture of3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)benzenesulfonamide (296.00mg, 935.73 μmol, 1.50 eq) and4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzoicacid (371.25 mg, 623.82 μmol, 1.00 eq) in THF (20.00 mL) was added EDCI(358.76 mg, 1.87 mmol, 3.00 eq) and DMAP (228.64 mg, 1.87 mmol, 3.00eq). The mixture was stirred at 25° C. for 12 h. After removal of thesolvent, the residue was purified together with EW6259-90 by Prep-HPLC(column: Phenomenex Synergi C,₈ 150*25*10 μm; mobile phase: [water(0.05%HCl)-ACN]; B%: 38%-58%, 7.8 min) to give4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((tetrahydro-2H-pyran-4-yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide(68.4 mg, 50.81 μmol, 8.15% yield) as yellow solid which was confirmedby LC-MS and ¹H NMR. LC-MS: EW6259-91-P1G (M+H⁺=893.2) 1HNMR:EW6259-91-P1P 400 MHz CD₃OD, δ 8.43 (s, 1 H), 8.22 (s, 1 H), 8.17 (d,J=2.0 Hz, 1 H), 8.01-8.04 (m, 1 H), 7.69-7.71 (m, 2 H), 7.33-7.39 (m, 3H), 7.24 (s, 1 H), 7.13-7.15 (m, 3 H), 6.64-6.65 (m, 1 H), 4.11-4.13 (m,2 H), 3.75-4.04 (m, 5 H), 3.75 (s, 2 H), 3.54-3.56 (m, 2 H), 3.48-3.51(m, 2 H), 3.31-3.41 (m, 2 H), 2.39 (s, 2 H), 2.14-2.17 (m, 3 H),1.80-1.83 (m, 2 H), 1.52-1.59 (m, 5 H), 1.03 (s, 6 H).

The compounds below are prepared by methods substantially identical,similar, or analogous to those disclosed in Scheme A-C and aboveexamples:

Ex- am- ple m/ No. Chemical Name z(MH⁺) 194-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 923biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoro-1-methylpiperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 204-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 910biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluoropiperidin-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 214-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 893biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((morpholin-2-ylmethyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 224-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 907biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-methylmorpholin-2-yl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 234-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 920biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-hydroxy-4-methylcyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 244-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 911biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-fluorotetrahydro-2H-pyran-4-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 254-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 921biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-hydroxy-4-methylcyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 26N-((3-chloro-4-((4-fluoro-1-(oxetan-3-yl)piperidin-4- 955yl)methoxy)phenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 27N-((3-chloro-4-((4-fluoro-1-methylpiperidin-4- 913yl)methoxy)phenyl)sulfonyl)-4-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)methyl)piperazin-1-yl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 284-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 961biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholinocyclohexyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 294-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 906biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-hydroxycyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 304-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 920biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4-methoxycyclohexyl)methyl)amino)-3- nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo [3,2- e]pyridin-1(5H)-yl)benzamide 314-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 941biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((1-(methylsulfonyl)pyrrolidin-3-yl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3 -b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 324-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 961biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3 -nitro-4-(((1-(tetrahydro-2H-pyran-4-yl)pyrrolidin-3-yl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 334-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 962biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-morpholinocyclohexyl)oxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 344-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 907biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-hydroxycyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 354-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 921biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-methoxycyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 364-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 921biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-hydroxy-4-methylcyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 374-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 942biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((1-(methylsulfonyl)pyrrolidin-3-yl)oxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 384-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 962biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((1-(tetrahydro-2H-pyran-4-yl)pyrrolidin-3-yl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 394-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 908biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4-methylmorpholin-2-yl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 404-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 926biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-(((4,4-difluorocyclohexyl)methyl)amino)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)- yl)benzamide 414-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 944biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4- (((1,4,4-trifluorocyclohexyl)methyl)amino)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 424-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 927biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((4-((4,4-difluorocyclohexyl)methoxy)-3-nitrophenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide 434-(4-((4′-chloro-5,5-dimethyl-3,4,5,6-tetrahydro-[1,1′- 945biphenyl]-2-yl)methyl)piperazin-1-yl)-N-((3-nitro-4-((1,4,4-trifluorocyclohexyl)methoxy)phenyl)sulfonyl)-2-(pyrazolo[4,3-b]pyrrolo[3,2-e]pyridin-1(5H)-yl)benzamide

BIOLOGICAL EXAMPLE 1 Bcl-2 Competition Binding (FluorescencePolarization) Assay

The fluorescence-labeled 23 amino acid peptide BH3 was purchased fromCalBiochem (NLWAAQRYGRELRRMSDKFVD). An unbound Fluorescein labeled BH3peptide emits random light with respect to the plane of polarizationplane of excited light, resulting in a lower polarization degree (mP)value. When the peptide is bound to Bcl-2, the complex tumble slower andthe emitted light can have a higher level of polarization, resulting ina higher mP value. This binding assay was performed in 96-well plate andwith each assay contained 15 and 30nM of labeled peptide and purifiedBcl-2 protein (purchased from R&D Systems, Inc). The assay buffercontained 20 mM Hepes (pH 7.0), 50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄,0.1 mg/ml Bovine Gamma Globulin and 0.01% NP40. Compounds were dilutedin DMSO and added to the final assay with concentration range from 20 uMto 2 nM. The polarization degree (mP) value was determined by BioTekSynergy II with background subtraction after 3 hours of incubation atroom temperature. IC₅₀ was calculated using Prism software withsigmoidal dose-response curve fitting. ABT-737 was used as referencecompound. Such assays, carried out with a range of doses of testcompounds, allow the determination of an approximate IC₅₀ value.Although the inhibitory properties of the compounds of the presentinvention vary with structural change as expected, the activitygenerally exhibited by these agents is in the range of IC₅₀=0.1-1000 nM.The following table lists the IC₅₀ values of certain compounds of theinvention.

Example No. BCL-2 ABT-199 <10 nM ABT-263 <10 nM 4 <10 nM 6 <10 nM 7 <10nM 8 <10 nM 12 <10 nM 13 <10 nM 16 <10 nM 11 <10 nM 14 <10 nM 17 <10 nM15 <10 nM 19 <10 nM 18 <10 nM

BIOLOGICAL EXAMPLE 2 In Vitro Anti-Proliferation Assay in BCL-2DependentAcute Lymphoblastic Leukemia Cell Line RS4; 11

Cell antiproliferation is assayed by PerkinElmer ATPlite™ LuminescenceAssay System. Briefly, the various test cancer cell lines are plated ata density of about 1×10⁴ cells per well in Costar 96-well plates, andare incubated with different concentrations of compounds for about 72hours in medium supplemented with 5% FBS. One lyophilized substratesolution vial is then reconstituted by adding 5 mL of substrate buffersolution, and is agitated gently until the solution is homogeneous.About 50 μL of mammalian cell lysis solution is added to 100 μL of cellsuspension per well of a microplate, and the plate is shaken for aboutfive minutes in an orbital shaker at ˜700 rpm. This procedure is used tolyse the cells and to stabilize the ATP. Next, 50 μL substrate solutionis added to the wells and microplate is shaken for five minutes in anorbital shaker at ˜700 rpm. Finally, the luminescence is measured by aPerkinElmer TopCount® Microplate Scintillation Counter. Such assays,carried out with a range of doses of test compounds, allow thedetermination of the cellular anti-antiproliferative IC₅₀ of thecompounds of the present invention. The following table lists the IC₅₀values of certain compounds of the invention.

Example No. RS4; 11 IC₅₀ 4 <15 nM 12 <15 nM

Biological Example 3 In Vitro Anti-Proliferation Assay in BCL-XLDependent Lines Small Cell Lung Cancer Cell Line H146

Cell antiproliferation is assayed by PerkinElmer ATPlite™ LuminescenceAssay System. Briefly, the various test cancer cell lines are plated ata density of about 1×10⁴ cells per well in Costar 96-well plates, andare incubated with different concentrations of compounds for about 72hours in medium supplemented with 5% FBS. One lyophilized substratesolution vial is then reconstituted by adding 5 mL of substrate buffersolution, and is agitated gently until the solution is homogeneous.About 50 μL of mammalian cell lysis solution is added to 100 μL of cellsuspension per well of a microplate, and the plate is shaken for aboutfive minutes in an orbital shaker at ˜700 rpm. This procedure is used tolyse the cells and to stabilize the ATP. Next, 50 μL substrate solutionis added to the wells and microplate is shaken for five minutes in anorbital shaker at ˜700 rpm. Finally, the luminescence is measured by aPerkinElmer TopCount® Microplate Scintillation Counter. Such assays,carried out with a range of doses of test compounds, allow thedetermination of the cellular anti-antiproliferative IC₅₀ of thecompounds of the present invention. The following table lists the IC50values of certain compounds of the invention in the BCL-XL depedent cellline H146, indicating that Example 4, like ABT-199, is a BCL-2 selectiveinhibitor.

Example No. H146 IC₅₀ (nM) ABT-263 60.0 ABT-199 >2000 4 >2000 12 >2000

BIOLOGICAL EXAMPLE 4 In Vivo Xenograft Studies

Typically, athymic nude mice (CD-1 nu/nu) or SCID mice are obtained atage 6-8 weeks from vendors and acclimated for a minimum 7-day period.The cancer cells are then implanted into the nude mice. Depending on thespecific tumor type, tumors are typically detectable about two weeksfollowing implantation. When tumor sizes reach 100-200 mm³, the animalswith appreciable tumor size and shape are randomly assigned into groupsof 8 mice each, including one vehicle control group and treatmentgroups. Dosing varies depending on the purpose and length of each study,which typically proceeds for about 3-4 weeks. Tumor sizes and bodyweight are typically measured three times per week. In addition to thedetermination of tumor size changes, the last tumor measurement is usedto generate the tumor size change ratio (T/C value), a standard metricdeveloped by the National Cancer Institute for xenograft tumorevaluation. In most cases, % T/C values are calculated using thefollowing formula: % T/C=100×ΔT/ΔC if ΔT>0. When tumor regressionoccurred (ΔT<0), however, the following formula is used: %T/T0=100×ΔT/T0. Values of <42% are considered significant.

What is claimed is:
 1. A compound of Formula (I), or an N-oxide thereof,or a pharmaceutically acceptable salt, solvate, polymorph, tautomer,stereoisomer, an isotopic form, or a prodrug of said compound of Formula(I) or N-oxide thereof:

wherein Z₁ is absent, (CH₂)_(p), N(H), O, S, C(O), S(O)₂, OC(O), C(O)O,OS(O)₂, S(O)₂O, C(O)S, SC(O), C(O)C(O), C(O)N(H), N(H)C(O), S(O)₂N(H),N(H)S(O)₂, OC(O)O, OC(O)S, OC(O)N(H), N(H)C(O)O, N(H)C(O)S,N(H)C(O)N(H), (CH₂)_(p)N(H)(CH₂)_(q), (CH₂)_(p)N(H)C(O)(CH₂)_(q),(CH₂)_(p)C(O)N(H)(CH₂)_(q), OC(O)N(H)(CH₂)_(p+1)N(H)(CH₂)_(q), abivalent alkenyl group, or a bivalent alkynyl group; W is CH or N, andthe tricylic

is optionally substituted with one or more R₇; each of R₁, R₂, R₃, R₄,R₅, R₆, and R₇, independently, is H, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,heteroaryl, halo, nitro, oxo, cyano, OR_(a), SR_(a), alkyl-R_(a),NH(CH₂)_(p)R_(a), C(O)R_(a), S(O)R_(a), SO₂R_(a), C(O)OR_(a),OC(O)R_(a), NR_(b)R_(c), P(O)R_(b)R_(c), C(O)N(R_(b))R_(c),N(R_(b))C(O)R_(c), SO₂N(R_(b))R_(c), or N(R_(b))SO₂R_(c), in which saidcycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl,heteroaryl is optionally substituted with one or more R_(d); optionally,two of R₂ group, taken together with the atom to which they areattached, may optionally form a cycloalkyl or heterocycloalkyloptionally substituted with one or more R_(d); R_(a), R_(b), R_(c) andR_(d), independently, is H, alkyl, alkenyl, alkynyl, halo, cyano, amine,nitro, hydroxy, C(O)NHOH, alkoxy, alkoxyalkyl, haloalkyl, hydroxyalkyl,aminoalkyl, alkylcarbonyl, alkoxycarbonyl, alkylcarbonylamino,alkylamino, oxo, halo-alkylamino, cycloalkyl, cycloalkenyl,heterocycloalkyl, heterocycloalkenyl, aryl, or heteroaryl; L is absent,alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene,heterocycloalkylene, heterocycloalkenylene, wherein said alkylene,alkenylene, alkynylene, cycloalkylene, cycloalkenylene,heterocycloalkylene, or heterocycloalkenylene is optionally substitutedwith one or more R_(d); and each of m, n, p, and q, independently, is 0,1, 2, 3, or
 4. 2. The compound according to claim 1 or an N-oxidethereof, or a pharmaceutically acceptable salt, solvate, polymorph,tautomer, stereoisomer, an isotopic form, or a prodrug thereof, whereinthe compound is represented by Formula (II)

wherein the tricylic

is unsubstituted.
 3. The compound according to claim 2 or an N-oxidethereof, or a pharmaceutically acceptable salt, solvate, polymorph,tautomer, stereoisomer, an isotopic form, or a prodrug thereof, whereinthe compound is represented by Formula (III)


4. The compound according to claim 3 or an N-oxide thereof, or apharmaceutically acceptable salt, solvate, polymorph, tautomer,stereoisomer, an isotopic form, or a prodrug thereof, wherein R₁ is Cl.5. The compound according to claim 4 or an N-oxide thereof, or apharmaceutically acceptable salt, solvate, polymorph, tautomer,stereoisomer, an isotopic form, or a prodrug thereof, wherein R₅independently, is nitro, halo, or SO₂R_(a).
 6. The compound according toclaim 5 or an N-oxide thereof, or a pharmaceutically acceptable salt,solvate, polymorph, tautomer, stereoisomer, an isotopic form, or aprodrug thereof, wherein Z₁ is absent, NH, O, or S.
 7. The compoundaccording to claim 6 or an N-oxide thereof, or a pharmaceuticallyacceptable salt, solvate, polymorph, tautomer, stereoisomer, an isotopicform, or a prodrug thereof, wherein L is absent or C₁₋₃alkylene.
 8. Thecompound according to claim 7 or an N-oxide thereof, or apharmaceutically acceptable salt, solvate, polymorph, tautomer,stereoisomer, an isotopic form, or a prodrug thereof, wherein R₆ is H,4-6 membered cycloalkyl or 4-6 membered heterocyclyl, wherein the 4-6membered cycloalkyl or 4-6 membered heterocyclyl is optionallysubstituted with one or more substituents selected from the groupconsisting of halo, C₁₋₄alkyl, CN, —OH, C₁₋₄alkoxy, —S(O)₂CH₃, —COCH₃,3-6 membered cycloalkyl, and 3-6 membered heterocyclyl,
 9. The compoundaccording to claim 1 or an N-oxide thereof, or a pharmaceuticallyacceptable salt, solvate, polymorph, tautomer, stereoisomer, an isotopicform, or a prodrug thereof, wherein the compound is


10. The compound according to claim 1 or an N-oxide thereof, or apharmaceutically acceptable salt, solvate, polymorph, tautomer,stereoisomer, an isotopic form, or a prodrug thereof, wherein thecompound is


11. A pharmaceutical composition comprising a compound of Formula (I) oran N-oxide thereof as defined in claim 1, or a pharmaceuticallyacceptable salt, solvate, polymorph, tautomer, stereoisomer, an isotopicform, or a prodrug of said compound of Formula (I) or an N-oxidethereof, and a pharmaceutically acceptable diluent or carrier.
 12. Amethod of treating a neoplastic disease or autoimmune disease,comprising administering to a subject in need thereof an effectiveamount of a compound of Formula (I) or an N-oxide thereof as defined inclaim 1, or a pharmaceutically acceptable salt, solvate, polymorph,tautomer, stereoisomer, an isotopic form, or a prodrug of said compoundof Formula (I) or an N-oxide thereof.
 13. The method according to claim12, wherein the neoplastic disease is leukemia, lymphoma, or multiplemyeloma.
 14. A compound or an N-oxide thereof, or a pharmaceuticallyacceptable salt, solvate, polymorph, tautomer, stereoisomer, an isotopicform, or a prodrug of said compound or N-oxide thereof, wherein thecompound is:


15. A pharmaceutical composition comprising a compound or an N-oxidethereof as defined in claim 14, or a pharmaceutically acceptable salt,solvate, polymorph, tautomer, stereoisomer, an isotopic form, or aprodrug of said compound or an N-oxide thereof, and a pharmaceuticallyacceptable diluent or carrier.
 16. A method of treating a neoplasticdisease or autoimmune disease, comprising administering to a subject inneed thereof an effective amount of a compound or an N-oxide thereof asdefined in claim 14, or a pharmaceutically acceptable salt, solvate,polymorph, tautomer, stereoisomer, an isotopic form, or a prodrug ofsaid compound or an N-oxide thereof.
 17. The method according to claim16, wherein the neoplastic disease is leukemia, lymphoma, or multiplemyeloma.